mu-Opioid receptor-expressing neurons in the rat cerebral neocortex were characterized by an immunolabeling method with an antibody to a carboxyl terminal portion of the receptor. They were small, bipolar, vertically elongated, non-pyramidal neurons, and scattered mainly in layers II-IV. We examined chemical characteristics of mu-opioid receptor-expressing neocortical neurons by the double immunofluorescence method. Almost all neuronal cell bodies expressing mu-opioid receptor-like immunoreactivity showed immunoreactivity for GABA, suggesting that they were cortical inhibitory interneurons. mu-Opioid receptor-immunoreactive neurons were further studied by the double staining method with markers for the subgroups of cortical GABAergic neurons. Immunoreactivities for vasoactive intestinal polypeptide, corticotropin releasing factor, choline acetyltransferase, calretinin and cholecystokinin were found in 92, 79, 67, 35 and 35% of mu-opioid receptor-immunoreactive cortical neurons, respectively. In contrast, less than 10% of mu-opioid receptor-immunoreactive neurons showed immunoreactivity for parvalbumin, calbindin, somatostatin, neuropeptide Y or nitric oxide synthase. Moreover, mu-opioid receptor-immunoreactive neurons very frequently exhibited preproenkephalin immunoreactivity, but not preprodynorphin immunoreactivity. The present results indicate that mu-opioid receptor-expressing neurons belong to a distinct subgroup of neocortical GABAergic neurons, because vasoactive intestinal polypeptide, corticotropin releasing factor, choline acetyltransferase, calretinin and cholecystokinin have often been reported to coexist with one another in single neocortical neurons. Methionine-enkephalin, which is a major product of the preproenkephalin gene, is known to be one of the most potent endogenous ligands for mu-opioid receptor. Thus, the expression of mu-opioid receptor in preproenkephalin-producing neurons suggested that mu-opioid receptor serves as an autoreceptor for the subpopulation of GABAergic interneurons at a single-neuron or population level.