Pre-embedding immunohistochemistry with monoclonal antibody to choline acetyltransferase was combined with post-embedding immunohistochemistry with antisera to GABA and glycine in order to study the pattern of coexistence of GABA, glycine and acetylcholine in neurons in lamina III of rat spinal dorsal horn. Of 50 neurons which were choline acetyltransferase immunoreactive, 47 showed GABA-like immunoreactivity and none were immunoreactive with antiserum to glycine, despite the fact that glycine is thought to be present in the majority of GABAergic neurons in lamina III. This suggests that while acetylcholine and glycine can both coexist with GABA in lamina III neurons, they are present in different populations of GABAergic cells. Taken together with recent ultrastructural evidence concerning the synaptic connections of glycinergic and cholinergic structures in the dorsal horn, this suggests that there are functional differences between neurons which contain GABA and glycine and those which contain GABA and acetylcholine.