Previous studies have indicated that nitric oxide, a labile freely diffusible biological messenger synthesized by nitric oxide synthase, may modulate light transduction and signal transmission in the retina. In the present work, the large size of retinal cells in tiger salamander (Ambystoma tigrinum) allowed the utilization of nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase histochemistry and nitric oxide synthase immunocytochemistry to delineate the cell-specific intracellular localization of nitric oxide synthase. NADPH-diaphorase activity was highly concentrated in the outer retina, in rod and cone inner segment ellipsoids, and between and adjacent to the photoreceptor cell bodies in the outer nuclear layer. Examination of enzymatically isolated retinal cells indicated that outer nuclear layer NADPH-diaphorase activity was localized to the distal processes of the retinal glial (Müller) cells and to putative bipolar cell Landolt clubs. Less intense NADPH-diaphorase activity was seen in the photoreceptor inner segment myoid region, in a small number of inner nuclear layer cells, in cap-like configurations at the distal poles of cells in the ganglion cell layer and surrounding ganglion cell layer somata, and in punctate form within both plexiform layers, the pigment epithelium, and the optic nerve. Nitric oxide synthase-like immunoreactivity was similarly localized, but was also concentrated along a thin sublamina centered within the inner plexiform layer. The potential for nitric oxide generation at multiple retinal sites suggests that this molecule may play a number of roles in the processing of visual information in the retina.