AbstractWe analyzed the distribution and light‐microscopic features of the NADPH diaphorase‐containing structures in the lizard hippocampus, likely to correspond to nitric oxide. We also studied co‐localization of NADPH diaphorase with the neurotransmitter GABA, the calcium‐binding protein parvalbumin, and the neuropeptide somatostatin, in order to examine whether putative nitric oxide‐synthesizing neurons represent a different subpopulation of GABA cells, on which the authors recently reported in lizards. We also studied co‐localization of NADPH diaphorase with parvalbumin or somatostatin in mice to ascertain whether the characteristics of this population in reptiles parallel the situation in mammals. Most of the positive NADPH diaphorase neurons were stained in a Golgi‐like manner and were in the plexiform layers of the lizard hippocampus with morphologies ranging from bipolar to multipolar. Co‐localization with GABA was 100%, and NADPH diaphorase‐positive neurons in the lizard hippocampus did not contain parvalbumin or somatostatin. The results indicate that putative nitric oxide‐synthesizing neurons represent a distinct subpopulation of GABA interneurons in the lizard hippocampus. Two different types of fibers were described in the plexiform layers: one type bearing thick varicosities, and the other thinner ones. We discuss the possibility that at least part of the positive fibers arise from a hypothalamic aminergic nucleus containing the third ventricle, the periventricular hypothalamic organ. Most radial glia were stained almost completely and formed typical end‐feet both at the pia and around capillaries. The results of this study confirm that the capacity for synthesizing nitric oxide is linked to a determined set of neuronal markers depending on the specific brain region, and they provide new resemblances between hippocampal regions in different classes of vertebrates.