Studies have shown that interneurons expressing neuronal nitric oxide synthase (nNOS) have a widespread distribution in the mouse brain and the density of nNOS-expressing interneurons is different among brain regions.The dentate gyrus (DG) of the hippocampus is known to contain higher density of nNOS-expressing interneurons, however issues regarding the laminar distribution, GABAergicity and the neurochemical profiles of those nNOS-expressing interneurons have not been characterized.In the present study, we investigated the characteristics of nNOS-expresing interneurons in the dentate gyrus of the rat by focusing on their laminar distribution, GABAergicity, and co-localization with other commonly known interneuron molecular markers.Experiments were conducted in adult male Sprague-Dawley rats (11-13 weeks old).nNOS-expressing cells and cells positive for another interneuron molecular markers were identified immunohistochemically and images were captured with a Zeiss LSM 780 confocal microscopy.Densities of immunoreactive positive cells (cells/mm2)were calculated by dividing the number of cells with the area in each layer.The results showed that within the DG,nNOS-expressing neurons were present in all three layers with the higher density to appear in the hilus.Co-labeling nNOS and glutamate decarboxylase 67 (GAD67) showed that near two-third of GABAergic interneurons were nNOS-positive also.The percentages of nNOS-positive cells that co-expressed GAD67 are laminar dependent;in the hilus, near 20% of nNOS-expressing cells lacked GAD67 expression.nNOS-positive cells that co-expressed other commonly known interneuron molecular markers including cholecystokinin (CCK), vasoactive intestinal polypeptide (VIP), parvalbumin (PV), somatostatin (SOM), neuropeptide Y (NPY) and calretinin (CR) ranged from 2% to 30%, respectively.In the molecular layer, co-labeling nNOS and one of above mentioned interneuron markers showed that the total numbers of nNOS-positive cells far exceeded the sum of cells expressing other commonly known interneuron molecular markers.We conclude that (1) nNOS-expressing cells account near twothird of GAGAergic interneurons in the rat dentate gyrus;(2) the distribution of nNOS-expressing neurons in the rat DG is laminar dependent, and (3) interneurons that express nNOS but not other commonly known interneuronal molecular markers do exist in the DG (at least the molecular layer).