In some brain regions, previous studies reported the frequent coexistence between neuronal nitric oxide synthase (nNOS) and somatostatin (SOM). In the hippocampus, nNOS and SOM were mainly expressed in GABAergic nonprincipal neurons. Here we estimated the immunocytochemical colocalization of nNOS and SOM in the mouse hippocampus using the optical disector. Both in the Ammon's horn and dentate gyrus, we encountered only a few nNOS-immunoreactive (IR)/SOM-like immunoreactive (LIR) neurons. They were mainly located in the stratum oriens of the Ammon's horn and in the dentate hilus. The nNOS-IR/SOM-LIR neurons usually showed characteristic large somata with thick dendrites, whereas the majority of nNOS-IR/SOM-negative neurons showed small somata with thin dendrites. Quantitative data revealed that the double-labeled cells represented only 4% and 7% of nNOS-IR neurons and SOM-LIR neurons, respectively, in the whole area of the hippocampus. We also found the laminar and dorsoventral differences in the degree of colocalization between nNOS and SOM. The percentages of nNOS-IR neurons containing SOM-like immunoreactivity were relatively high in the stratum oriens of the ventral CA1 region (24%), stratum lucidum of the dorsal CA3 region (29%) and dorsal dentate hilus (32%), but they were quite low in the other layers. On the other hand, the percentages of SOM-LIR neurons containing nNOS immunoreactivity were somewhat high in the stratum lucidum of the dorsal CA3 region (19%) and dorsal dentate hilus (28%), whereas they were very low in the other layers. Immunofluorescent triple labeling of axon terminals for nNOS, SOM and glutamic acid decarboxylase indicated that some nNOS-IR/SOM-LIR neurons might be dendritic inhibitory cells. The present results show the infrequent colocalization of nNOS and SOM in the mouse hippocampus, and also suggest that the double-labeled cells may be a particular subpopulation of hippocampal GABAergic nonprincipal neurons.