Light sensitivity in the vertebrate retina is mediated by the opsin visual pigments inside rod and cone photoreceptor cells. These pigments consist of a G protein-coupled receptor and the photo-sensitive ligand, 11-cis-retinaldehyde (11-cis-RAL). Absorption of a photon by an opsin pigment induces isomerization of the 11-cis-RAL chromophore to all-trans-retinaldehyde (all-trans-RAL), rendering the pigment insensitive to light. The bleached opsin regains light sensitivity by recombining with another 11-cis-RAL. The vertebrate eye contains a biochemical mechanism for regenerating 11-cis-RAL chromophore from all-trans-RAL, called the visual cycle. The visual cycle takes place within cells of the retinal pigment epithelium (RPE). A second visual cycle also appears to be present in Müller glial cells of the retina. A critical step in the regeneration of 11-cis-RAL chromophore is thermal re-isomerization to the 11-cis configuration of an all-trans-retinyl ester (all-trans-RE) or an all-trans-retinol (all-trans-ROL). In RPE cells, this step is carried out by an enzyme called Rpe65 isomerase. This chapter provides methods for assaying Rpe65 isomerase. Although Rpe65 utilizes an all-trans-RE such as all-trans-retinyl palmitate (all-trans-RP) as substrate, it can be assayed in RPE homogenates by providing all-trans-ROL substrate and allowing the endogenous lecithin:retinol acyl transferase (LRAT) to synthesize all-trans-REs using fatty acids from phosphatidylcholine in the membranes. Alternatively, all-trans-RP can be provided directly as substrate, although this requires the isomerase reaction to be carried out in the presence of detergent, since fatty-acyl esters of all-trans-ROL are insoluble. Methods are provided in this chapter for assaying Rpe65 in RPE homogenates with both all-trans-ROL and all-trans-RP substrates. A second visual cycle appears to be present in the retinas of cone-dominant species such as chicken. This retinal pathway may augment the RPE to provide 11-cis-RAL to cone photoreceptors under conditions of bright light where the rate of opsin photoisomerization is high. The isomerase in this pathway (isomerase-2) utilizes all-trans-ROL and palmitoyl coenzyme A (palm CoA) as substrates to synthesize 11-cis-retinyl palmitate (11-cis-RP). Isomerase-2 appears to be present in Müller cells but has not yet been identified. Methods are provided in this chapter for assaying isomerase-2 in chicken retina homogenates.