Inhibitory glycine receptors (GlyRs) are known to mediate postsynaptic inhibition in spinal cord, brain stem and some higher brain regions. Several developmentally and regionally regulated GlyR isoforms exist, which result from a differential expression of the GlyR alpha (alpha1-alpha4) and beta subunit genes. Currently, very little is known about GlyRs containing the alpha4 subunit, whose existence was predicted from a partial genomic sequence. Here, we describe the isolation of complementary DNA (cDNA) sequences for the mouse and chick GlyR alpha4 subunits. We show that a mouse GlyR alpha4 subunit full-length cDNA directs the formation of functional homo-oligomeric strychnine-sensitive GlyRs in Xenopus laevis oocytes and mammalian cells, and that these resemble GlyRs composed of the alpha1 subunit in pharmacological profile and single-channel properties. In situ hybridization reveals high levels of GlyR alpha4 subunit transcripts in the embryonic (E13) chick spinal cord, lumbosacral sympathetic ganglia and dorsal root ganglia. The avian GlyR alpha4 subunit gene also shows male-specific expression in the developing genital ridge. The pharmacological profile of alpha4 subunit-containing receptors and deduced location of the avian GlyR alpha4 subunit are consistent with it being a component of the embryonic excitatory GlyRs previously identified in sympathetic neurons. Our data also suggest a novel role for GlyRs in the maturation of reproductive organs.