Glycine receptors (GlyRs) are pentameric ligand-gated ion channels (pLGICs) that mediate fast synaptic transmission. A GlyR subunit comprised of a large N-terminal extracellular domain (ECD), four transmembranedomains (TMD1–4), a long intracellular loop (IL) connecting M3 and M4, and a short extracellular C-terminus. At the first glance, one would suppose that the primary task of the TMDs is to anchor the protein in the cell membrane. However, TMDs are more than just anchors: They build in their entirety the central located ion conducting channel and mutations of TMD amino acids can result in massive functional disorders. Moreover, previously published data indicate that structural rearrangements between the TMDs exist, which are key processes for the opening and closing of the channel. Therefore, it is of great interest to increase the knowledge about the TMDs functions because a decreased GlyR activity is evident in chronic inflammatory and neuropathic pain as well as in the hyperekplexia disease. In this study we characterized the role of the GlyR TMDs for the assembly, function and allosteric modulation by the general anesthetic propofol (pro) and derivates. We found that the TMDs are important determinants for the assembly and function of both the α1 Gly- and the 5-HT3A receptor. We also characterized the role of the TMDs for the allosteric modulation of the GlyR function by the general anesthetic propofol (pro) and derivates.