In the classic retinoid cycle, 11-cis retinol is synthesized in the retinal pigment epithelium (RPE) by two enzymes: Isomerase I (RPE65) and lecithin:retinol acyltransferase (LRAT). The purpose of this study is to provide experimental evidence for two active isomerases in the cone-dominated chicken eye: an LRAT-dependent Isomerase I in the RPE and an ARAT (acyl CoA:retinol acyltransferase)-dependent isomerase (Isomerase II) in the retina. First, we show that whole chicken retina in vitro, removed from the RPE/choroid and sclera, produces 11-cis retinoids upon light exposure, indicating the existence of RPE-independent isomerase (Isomerase II) activity in the retina. RT-PCR studies show high levels of RPE65 expression in the RPE, low levels in the retina, and none in primary Müller cell cultures, indicating the presence of Isomerase I in the RPE and a minimal amount in the retina. Activities of the RPE and retina isomerases were then measured by enzyme assays with specific enzyme inhibitors. 2,2′-Bipyridine, a known Isomerase I inhibitor, and N-ethyl-maleimide (NEM), a known LRAT inhibitor, significantly reduced Isomerase I activity but not Isomerase II activity. Progesterone, a known ARAT inhibitor, completely blocked Isomerase II activity but not Isomerase I activity. Thus the present study reports novel results to distinguish the biochemical properties of Isomerase I from Isomerase II, as well a difference in their locations in the chicken eye. Based on these differences, the cone-dominated chicken eye must contain two retinoid cycles: a classic visual cycle for retinoid exchange between the RPE and the retina supported by Isomerase I in the RPE, and an additional visual cycle for retinoid processing in the retina supported by Isomerase II.Light sensitivity in the eye requires the regeneration of 11-cis retinaldehyde through different isomers and oxidation states of vitamin A. An intimate relationship exists between photoreceptors and the retinal pigment epithelium (RPE) to regenerate this 11-cis retinaldehyde by way of the biochemical pathway known as the visual or retinoid cycle. This classic retinoid cycle has been well established for rod photoreceptors (for review see (1-7)). Attention has recently been focused on an additional vitamin A processing pathway in the retina for the supply of visual chromophore to cone photoreceptors (6;8-13). Little is known about the pathway of this novel cone cycle (6).A key reaction in both retinoid cycles involves the isomerization of retinoids from the alltrans to the 11-cis conformation. The first (and more extensively characterized) pathway is enzymatic and driven by the protein RPE65 (Isomerase I) (14-16) where it is localized in the RPE and uses all-trans retinyl esters as the substrate for the production of 11-cis retinol (17; 18). RPE65 from the RPE of the cone-dominated chicken has been shown to possess significantly higher isomerase activity than the RPE from rod-dominated species (12).The second pathway is a photic cycle (5) driven by the retinal G prote...