The increase in cyclic GMP contents in phototransduction is likely to be mediated with release of nitric oxide (NO). In this study we have characterized a constitutive NO synthase (NOS) isolated from rat retina supernatant. The activity of NOS was determined by monitoring L-citrulline formation from L-arginine. Soluble NOS from retina used L-arginine as substrate, with NADPH, tetrahydrobiopterin and FAD as cofactors. The enzyme activity was raised with additional calcium and was reduced with high concentrations of calmodulin inhibitors. Protein immunoblot and immunohistochemical analysis using a specific antibody against Type I NOS indicated that the same type of NOS is mainly localized in amacrine cells in the retina. However, the enzyme activity in freshly prepared sample was not completely abolished in the absence of calcium or with calmodulin inhibitors. In addition, NADPH diaphorase staining in retina was wider and more intensive than staining with the antibody against Type I NOS. These results indicate that rat retina contains more than one type of NOS.