Purpose. Phototransduction in cones is initiated by the bleaching of their visual pigment, which comprises a protein component—cone opsin—and a vitamin A derivative—11-cis retinal. Little is known about the source of 11-cis retinal for cones. In the current study, neural retina leucine zipper–deficient (Nrl−/−) and rod opsin (Rho−/−)–deficient mice were used, two mouse models that have been described as having a “cone-only” retina, to analyze the retinoid metabolism of cones. In addition, these mice were bred to retinal pigment epithelial protein 65 (Rpe65−/−)–deficient mice to study the role of RPE65. methods. Mice were analyzed using morphology, Western blot analysis, immunohistochemistry, electroretinography (ERG), and retinoid profiling by HPLC. results. In comparison to wild-type mice, the retina of Nrl−/− mice contained elevated levels of RPE65 and cellular retinaldehyde-binding protein (CRALBP), suggesting a particular role of these two proteins for the retinoid metabolism of cones. In Nrl−/− mice, different retinoid species were present in proportions similar to wild type. Ablation of RPE65 in Nrl−/− and Rho−/− mice led to the absence of 11-cis retinal, but increased the total retinoid content, with retinyl esters representing the most abundant retinoid species. In the absence of RPE65, retinal sensitivity in Nrl−/− mice dropped by a factor of a thousand. conclusions. The data show that RPE65, previously shown to be essential for rod function, is also indispensable for the production of 11-cis retinal for cones and thus for cone function.