Robert Koch already described experiments where he used Tuberculin for intradermal injec-tion in guinea pigs. Based on this work Clemens von Pirquet developed the Tuberculin Skin Test (TST) which is still used today for the diagnosis of tuberculosis. Tuberculins are used in vitro and in vivo. Tuberculin is an antigen preparation obtained from the supernatant of mycobacterial cultures. During the manufacturing process harvested supernatant is heat inactivated via steam, cleared and protein enriched by filtration or acid precipitation. Phenol is allowed to be added as a preserving agent. Tuberculins are produced either from Mycobacterium tuberculosis culture for human use or from Mycobacterium bovis culture for bovine use. Different Tuberculins are defined according to the Mycobacteria strain they are based on. The manufacturing process includes different empiric steps that are hard to standardize. So every single Tuberculin batch has to be tested before market release. So far Tuberculins have been tested in animal experiments by using guinea pigs. The current test procedures according to the European Pharmacopoeia (Ph. Eur.) rely on the sensitization of guinea pigs either with live or inactivated mycobacteria. Not less than four weeks later the flanks are shaved and different dilutions of a reference preparation and the test batch of tuberculin are injected intradermally. Doses are chosen to produce lesions ranging from 8 – 25 mm. Diameters are measured and the potency of the test batch is calculated by regression analysis. As a result of statistical analysis a dose response curve is obtained and the potency of the test tuberculin in relation to the reference tuberculin is determined. According to § 2 and § 32 of the Tierimpfstoff-Verordnung the Paul-Ehrlich-Institute as Fed-eral Institute for Vaccines and Biomedicines is the responsible institution in the federal republic of Germany for the state-controlled batch potency testing of tuberculin. Due to the heat treatment, which is the crucial inactivation step during the manufacturing process, the majority of tuberculin antigens are denatured. This impedes potency deter-mination by molecular biological methods and is one of the reasons why tuberculins are still tested in an animal experiment. However, the animal test suffers from several drawbacks including the subjective read-out, the spatial limitations that only allow for few test replicates and/or dilutions, the poor reproducibility and a significant distress for the laboratory animals through the induction of painful, sometimes necrotizing lesions. Therefore we aimed to establish a new method for batch potency testing of bovine Tuberculin. The new assay is based on the direct measurement of tuberculin-specific T cell proliferation because the cellu-lar response is the underlying cause for the type IV hypersensitivity reaction measured in the skin test. The in vitro proliferation assay is developed in accordance to the 3R con-cept of Russel and Burch and shall disburden laboratory animals during batch potency testing of Tuberculins. The lethal infection with virulent Mycobacterium bovis is replaced with another sensitization strategy by using inactivated wet mass of Mycobacteria bovis strain AN5. It was the aim to replace the most distress part of the animal experiment – the Tubercu-lin Skin Test, refine the method by using flow cytometry as an objective read-out. For the new established method seven guinea pigs are sensitized with inactivated wet mass of Mycobacterium bovis strain AN5. On day 30 blood is obtained and Peripheral Blood Mon-onuclear Cells (PBMC) are purified via Ficoll gradient centrifugation. PBMCs are stained with Carboxyfluoresceinsuccimidylester (CFSE) and stimulated with Tuberculin in eight different dilutions. The response to a test batch is compared to a standard preparation. After 5 days CFSE dilution, as a measure for cell proliferation, is determined by flow cytometry. Due to the complexity of the underlying immunological processes, the variability of the test results – in particular the differences between individual animals of one group – was relatively high with both methods. This is an observation that is frequently made with biological potency assays. “However, in a direct comparison the proliferation assay was clearly more precise than the skin test. As this may indicate that the new assay yields more reliable test results, it is also important with respect to animal welfare, because it can help to avoid test repeats. Another aspect is the spatial limitations of the current test, which requires at least eight guinea pigs per test batch. With the proliferation assay a significant reduction of animal numbers can be achieved, because the format allows to test several batches in parallel. With this, the alternative approach is in accordance with the 3R concept proposed by Russel and Burch”. The data presented in this thesis allow for the conclusion that the proliferation assay holds promise to replace the current potency assay for bovine Tuberculins. Another aspect of the current thesis was to establish the techniques to study cellular immune responses of guinea pigs in general. The particular focus was on responses to defined my-cobacterial lipid antigens. Since T cells recognize mycobacterial lipid antigens in the context of group I CD1 molecules it was necessary to generate CD1 positive antigen presenting cells (APCs) in vitro to detect CD1 restricted T cells. To this end guinea pig cytokines were recombinantly expressed and used to stimulate guinea pig monocytes to express CD1. Sub-sequently, the APCs were used to stimulate lipid-specific T cells. Using this approach it could be demonstrated, that guinea pigs mount CD1 restricted T cell responses to Lipoarabi-nomannan (ManLAM) and Glucosemonomycolate (GMM) to a similar extend as observed in humans.