ABSTRACTNitric oxide (NO) synthesis in the retina is triggered by light stimulation. NO has been shown to modulate visual signal processing at multiple sites in the vertebrate retina, via activation of the most sensitive target of NO signaling, soluble guanylate cyclase. NO can also alter protein structure and function and exert biological effects directly by binding to free thiol groups of cysteine residues in a chemical reaction called S‐nitrosylation. However, in the central nervous system, including the retina, this reaction has not been considered to be significant under physiological conditions. Here we provide immunohistochemical evidence for extensive S‐nitrosylation that takes place in the goldfish and mouse retinas under physiologically relevant light intensities, in an intensity‐dependent manner, with a strikingly similar pattern in both species. Pretreatment with N‐ethylmaleimide (NEM), which occludes S‐nitrosylation, or with 1‐(2‐trifluromethylphenyl)imidazole (TRIM), an inhibitor of neuronal NO synthase, eliminated the light‐evoked increase in S‐nitrosylated protein immunofluorescence (SNI) in the retinas of both species. Similarly, light did not increase SNI, above basal levels, in retinas of transgenic mice lacking neuronal NO synthase. Qualitative analysis of the light‐adapted mouse retina with mass spectrometry revealed more than 300 proteins that were S‐nitrosylated upon illumination, many of which are known to participate directly in retinal signal processing. Our data strongly suggest that in the retina light‐evoked NO production leads to extensive S‐nitrosylation and that this process is a significant posttranslational modification affecting a wide range of proteins under physiological conditions. J. Comp. Neurol. 523:2082–2110, 2015. © 2015 Wiley Periodicals, Inc.