Cones recover their photosensitivity faster than rods after bleaching. It has been suggested that a higher rate regeneration of 11-cis-retinal, the chromophore for visual pigments, is required for cones to continuously function under bright light conditions. RPE65 is the isomerohydrolase catalyzing a key step in regeneration of 11-cis-retinal. The present study investigated whether RPE65 in a cone-dominant species is more efficient in its enzymatic activity than that from roddominant species. In vitro isomerohydrolase activity assay showed that isomerohydrolase activity in the chicken retinal pigment epithelium (RPE) was 11.7-fold higher than in the bovine RPE, after normalization by RPE65 protein levels. Similar to that of human and bovine, the isomerohydrolase activity in chicken RPE was blocked by two specific inhibitors of lecithin retinal acyltransferase, indicating that chicken RPE65 also uses all-trans-retinyl ester as the direct substrate. To exclude the possibility that the higher isomerohydrolase activity in the chicken RPE could arise from another unknown isomerohydrolase, we expressed chicken and human RPE65 using the adenovirus system in a stable cell line expressing lecithin retinal acyltransferase. Under the same conditions, isomerohydrolase activity of recombinant chicken RPE65 was 7.7-fold higher than that of recombinant human RPE65, after normalization by RPE65 levels. This study demonstrates that RPE65 from the cone-dominant chicken RPE possesses significantly higher specific retinol isomerohydrolase activity, when compared with RPE65 from rod-dominant species, consistent with the faster regeneration rates of visual pigments in cone-dominant retinas.