The distribution and extent of glutamate decarboxylase 65 (GAD65) mRNA-labeled neurons that coexpress pre-prosomatostatin mRNA were studied in the rat dentate gyrus of the dorsal and ventral hippocampal formation. The distribution of each group of neurons was determined initially by nonradioactive in situ hybridization experiments with digoxigenin-labeled riboprobes for GAD65 mRNA and pre-prosomatostatin mRNA. Double labeling experiments were then conducted with digoxigenin-labeled riboprobes for GAD65 mRNA and 35S-labeled riboprobes for pre-prosomatostatin mRNA. In the dorsal and ventral dentate gyrus, GAD65 mRNA-containing neurons were highly concentrated in the hilus and in the innermost part of the granule cell layer whereas only a few labeled neurons were scattered in the molecular layer. Pre-prosomatostatin mRNA-containing neurons were primarily located in the hilus and were virtually absent from the molecular and granule cell layers. The simultaneous detection of GAD65 and pre-prosomatostatin mRNAs in the same sections showed that the vast majority of pre-prosomatostatin mRNA-containing neurons in the hilus of the dentate gyrus were also labeled for GAD65 mRNA. In contrast many GAD65 mRNA-labeled neurons did not contain pre-prosomatostatin mRNA. These included all neurons in the molecular layer, neurons within the inner granule cell layer and neurons interspersed amongst double labeled neurons in the hilus. Quantitative analyses indicated that a very high percentage of hilar pre-prosomatostatin mRNA-containing neurons coexpressed GAD65 mRNA in the dorsal (96%) and ventral (92%) dentate gyrus. In contrast only a part of the total population of hilar GAD65 mRNA-containing neurons were also labeled for pre-prosomatostatin mRNA in the dorsal (43%) and ventral (53%) dentate gyrus. In the CA3c region, the percentages of neurons containing both mRNAs were similar to those observed in the hilus. The findings demonstrate that the vast majority of hilar somatostatin neurons, which have previously been shown to be extremely vulnerable to ischemia and seizure-induced damage, are GABA neurons. However, the total population of GAD65 mRNA-containing neurons in the hilus is substantially larger than the somatostatin-containing subgroup, and these findings reinforce the suggestion that GABA neurons are a major component of the diverse group of neurons in the hilus of the dentate gyrus.