In order to provide information about the function of glycine in the superficial three laminae of the rat dorsal horn and the possible coexistence of glycine and GABA at synapses in this region, we have carried out a combined study involving preembedding immunocytochemistry with a monoclonal antibody to the glycine receptor and postembedding immunocytochemistry with antiserum to fixed GABA. Glycine receptor-like immunoreactivity was present at axodendritic and axosomatic synapses in all three laminae, and at dendrodendritic synapses in lamina II. Although axons that formed axoaxonic synapses were often presynaptic at immunoreactive axodendritic synapses (and thus probably contained glycine), the axoaxonic synapses themselves did not show glycine receptor-like immunoreactivity. Many of the profiles that were presynaptic at glycine receptor-immunoreactive synapses showed GABA- like immunoreactivity. These results suggest that glycine acts as a postsynaptic inhibitory transmitter at various types of synapses in laminae I-III, and that it may coexist with GABA at many synapses in this region. However, it appears that while glycine and GABA may both be released at axoaxonic synapses, either glycine does not act as a transmitter at these synapses, or else it acts at an atypical receptor that was not recognized by the antibody used in this study.