In order to determine whether or not neuropeptide Y coexists with GABA or glycine in rat dorsal horn, we have examined 84 neuropeptide Y-immunoreactive neurons in laminae I-III with a combined pre- and postembedding immunocytochemical method. All of the neuropeptide Y-immuno-reactive neurons were also GABA-immunoreactive, but they were either non-immunoreactive or weakly immunoreactive with the glycine antiserum. In addition, a double-label immunofluorescence method was used to search for co-localization of neuropeptide Y and [Met]enkephalin in spinal cord. Although the two types of peptide immunoreactivity often coexisted in varicosities around the central canal and in the ventral horn, such coexistence was not seen in the superficial dorsal horn. These results suggest that neuropeptide Y is present in GABAergic neurons in laminae I-III of rat dorsal horn, but that it is largely or completely restricted to those neurons which do not contain glycine. In addition, the cells that contain GABA and neuropeptide Y appear to form a different population from those that contain GABA and [Met]enkephalin. Neuropeptide Y administered by intrathecal injection causes analgesia, and there is evidence that this may involve a presynaptic mechanism. The results of the present study suggest that neuropeptide Y may act in conjunction with GABA to produce presynaptic inhibition of nociceptive primary afferents.