Although glycine receptors are widely expressed in the forebrain their function is obscure. We studied their activation by two possible endogenous ligands, glycine and taurine, and demonstrate a different expression pattern of glycine receptors in neostriatal cholinergic interneurons from two rodent species. Single-cell-reverse transcription-polymerase chain reaction analysis of glycine receptor-subunit expression was combined with whole-cell recordings from acutely isolated cholinergic interneurons. All cells expressed the alpha2-glycine receptor subunit, the majority (72%) in mice but none in young and aged rats expressed the alpha3-subunit. The beta-subunit expression was associated with both a higher efficacy and a higher potency of the partial agonist taurine. Cells expressing the alpha3-subunit displayed a slower desensitization of taurine responses than of glycine responses, in contrast to cells expressing the alpha2-, beta-subunits where desensitization time constants were similar. Glycine responses were reduced by preapplication of taurine; this effect was more pronounced in cells lacking the alpha3-subunit. We demonstrate interspecies differences and heterogeneity in expression and function of glycine receptors within the same neuronal population in the neostriatum.