Hippocampal neurons containing GABA-, cholecystokinin(CCK)-, vasoactive intestinal polypeptide(VIP)-, or somatostatin(SS)-like immunoreactivity (LI) were localized in sections of rat hippocampus. GABA-, CCK-, VIP, and SS-LI are found exclusively in interneurons of the area dentata and hippocampus. In the area dentata, GABA-LI occurs in cells of all strata but predominates in type 1 and 2 basket cells. CCK-LI is present in a subset of these basket cells and some hilar cells. VIP-LI is present in a distinct subset of dentate interneurons that, unlike the type 1 and 2 basket cells, do not contribute to the fiber plexus in the inner molecular layer. These VIP-LI interneurons send their axons to nearby granule cells and form a plexus in the hilus. SS-LI, although rare in cells of the molecular and granular layers, is present in a large population of hilar interneurons that do not exhibit GABA-, CCK-, or VIP-LI. In area CA3 of the hippocampus, a variety of morphologically diverse interneurons containing GABA-, CCK-, VIP-, or SS-LI are present in all strata. In area CA1, SS-LI is present mainly in cells of strata oriens and pyramidale. GABA- CCK- and VIP-LI interneurons are present in all strata of CA1 but, unlike the SS-LI cells, are most numerous in strata pyramidale and radiatum. These findings in the area dentata, taken together with those of Kosaka et al. (J. Comp. Neurol. 239:967-969, '85), indicate that two main populations of interneurons can be discriminated on the basis of the substances they contain. One is a group of GABA-LI cells, some of which also contain CCK- and/or VIP-LI. These cells innervate the granule cells and the second group of interneurons, the SS-LI hilar cells, which apparently form part of the dentate ipsilateral associational/commissural projections.