In order to determine whether somatostatin coexists with GABA or glycine in neurones in rat spinal dorsal horn, a combined pre- and post-embedding immunocytochemical study was carried out. One hundred six somatostatin-immunoreactive neurones located in lamina II and the dorsal half of lamina III were tested with antiserum or monoclonal antibody to GABA and none of these cells showed GABA-like immunoreactivity. However, 8 out of 13 somatostatin-immunoreactive neurones located deeper in the dorsal horn (ventral lamina III and lamina IV) showed glycine-like immunoreactivity, and 6 of these were also GABA-immunoreactive. We have previously shown that neurotensin-immunoreactive neurones in laminae II and III are also not immunoreactive when tested with GABA antiserum (Todd et al.: Neuroscience 47:685-691, 1992), and a double-labelling fluorescence method was therefore used to compare the distribution of somatostatin and neurotensin within the superficial dorsal horn. The two types of peptide-immunoreactivity were never found in the same profile. These results suggest that somatostatin and neurotensin are present in different populations of non-GABAergic neurones in rat superficial dorsal horn, but that some somatostatin-containing neurones in the deeper part of the dorsal horn contain glycine, with or without GABA.