To date there are few compounds known to pharmacologically discriminate between alpha1 and alpha3 subunit-containing glycine receptors (GlyRs). The present study stemmed from an observation that the glycinergic agonists, taurine and beta-alanine, act with much lower agonist efficacy at alpha3 GlyRs than at alpha1 GlyRs. We sought to understand the structural basis of this difference to provide insights relevant to the development of alpha3-specific modulators as leads for the development of new anti-inflammatory analgesics. Using chimeras of alpha1 and alpha3 subunits, we identified the structurally divergent M4 transmembrane segment and C-terminal tail as a specific determinant of the efficacy difference. Because mutation of individual non-conserved M4 residues had little influence on agonist efficacies, the reduced agonist efficacy at alpha3 GlyRs is most likely a distributed effect of all non-conserved M4 residues. Given the lack of contact between M4 and other transmembrane segments, the efficacy differences are probably mediated by differential interactions with the surrounding lipid environment. This may explain why GlyR agonist efficacies differ among expression systems where membrane lipid composition is not conserved. It may also explain why GlyR agonist efficacy increases at high expression densities, as this would increase the propensity of receptors to cluster thereby inducing M4 segments of neighboring receptors to interact. This strong influence of M4 primary structure on partial agonist efficacy suggests that the relatively poorly conserved alpha3 GlyR M4 segment may be a promising domain to target in the search for alpha3 GlyR-specific modulators.