Regeneration of the visual chromophore, 11-cis-retinal, is a critical step in restoring photoreceptors to their dark-adapted conditions. This regeneration process, called the retinoid cycle, takes place in the photoreceptor outer segments and the retinal pigment epithelium (RPE). Disabling mutations in nearly all of the retinoid cycle genes are linked to human conditions that cause congenital or progressive defects in vision. Several mouse models with disrupted genes related to this cycle contain abnormal fatty acid retinyl ester levels in the RPE. To investigate the mechanisms of retinyl ester accumulation, we generated single or double knockout mice lacking retinoid cycle genes. Alltrans-retinyl esters accumulated in mice lacking RPE65, but they are reduced in double knockout mice also lacking opsin, suggesting a connection between visual pigment regeneration and the retinoid cycle. Only Rdh5-deficient mice accumulate cis-retinyl esters, regardless of the simultaneous disruption of RPE65, opsin, and prRDH. 13-cis-Retinoids are produced at higher levels when the flow of retinoid through the cycle was increased, and these esters are stored in specific structures called retinosomes. Most importantly, retinylamine, a specific and effective inhibitor of the 11-cisretinol formation, also inhibits the production of 13-cis-retinyl esters. The data presented here support the idea that 13-cis-retinyl esters are formed through an aberrant enzymatic isomerization process.Vitamin A transformations in the eye are essential for vision. Absorption of light by the vitamin A-derived chromophore of visual pigments, 11-cis-retinal, leads to its photoisomerization to all-trans-retinal and the initiation of the signal transduction cascade (1-4). Through a chain of reactions, all-trans-retinal is recycled enzymatically back to 11-cis-retinal (5-8). These retinoid cycle reactions (Figure 1) take place in the outer segments of photoreceptor cells and in the adjacent retinal pigment epithelium (RPE). 1 Characterization of the knockout mice lacking genes involved in the retinoid cycle provides important insight into the flow of retinoids in the eye.11-cis-Retinol dehydrogenase (RDH), also known as RDH5, catalyzes the final oxidation reaction of 11-cis-retinol in the RPE (9, 10). Although RDH5 is responsible for the majority of 11-cis-RDH activity, RDH11 and other RDHs also have a measurable role in regenerating the visual pigment by complementing RDH5 in RPE cells (11). Disruption of the gene encoding RDH5 causes fundus albipunctatus in humans (12,13). Fundus albipunctatus is an autosomal recessive form of congenital stationary night blindness characterized by the appearance of numerous small white dots located in the RPE, a delayed course of dark adaptation, and † This research was supported by NIH Grant EY09339. Lipid droplet-like organelles known as retinosomes were characterized as specific sites of alltrans-retinyl ester accumulation in the RPE (19,20). All-trans-retinyl esters accumulate in the RPE of wild-type mice as the...