Shuttling of retinoids across the interphotoreceptor matrix (IPM), 1 the extracellular space separating between retinal pigment epithelium (RPE) and photoreceptor cells in the eye, is essential for the function of the visual cycle. Rhodopsin regeneration requires transport of the visual chromophore 11-cisretinal from its site of synthesis in the RPE to photoreceptor cells. On exposure to light, rhodopsin-bound 11-cis-retinal is isomerized to all-trans-retinal, hydrolyzed from the protein, and is enzymatically reduced to all-trans-retinol. Regeneration of the visual chromophore requires movement of all-trans-retinol from photoreceptors back to the RPE for re-oxidation and isomerization (reviewed in Ref. 1). Thus, the visual cycle critically includes continuous transfer of all-trans-retinol from the photoreceptors to the RPE and of 11-cis-retinal from RPE to the neural retina. The mechanisms by which retinoids, which are poorly soluble in water, are transported through the IPM at rates that are sufficient to support visual function are not completely clear at present. It has been repeatedly suggested, however, that the interphotoreceptor retinoid-binding protein (IRBP), plays an important role in this process (reviewed in Ref.2).IRBP is a 140-kDa glycoprotein that is the major soluble protein in the IPM. It binds various isomeric and chemical forms of retinoids as well as long chain fatty acids (3-6), and is believed to mediate transfer of retinoids through the IPM (3, 7-16). IRBP possesses two retinoid-binding sites (3,8,17) that seem to be functionally distinct. The specific function of one of these sites (termed "site 1" in this article) is not clear at present but it has been shown to possess a retinoid selectivity in the order of 11-cis-retinal Ͼ all-trans-retinol Ͼ all-trans-retinal Ͼ 11-cis-retinol. The higher binding affinities for 11-cis-retinal and all-trans-retinol correspond to the physiological need to shuttle these particular retinoids across the IPM (18). It is thus conceivable that site 1 serves mainly to allow IRBP to maintain a large storage pool of these retinoid forms in the extracellular space of the IPM.The recent observations that the interactions of 11-cis-retinal with the other retinoid-binding site of IRBP ("site 2") are tightly regulated by the polyunsaturated long chain fatty acid docosahexaenoic acid (DHA), provided clues regarding the possible function of site 2. It was shown that binding of DHA to IRBP dramatically reduces the affinity of site 2 for this ligand, and facilitates the rate by which 11-cis-retinal is released from this site by about an order of magnitude (6). In contrast, DHA has very little effect on the interactions of 11-cis-retinal with site 1, or on the affinities of either site 1 or site 2 for all-transretinol (6). Further evidence indicated that DHA comprises a significant fraction of fatty acids associated with IRBP in the IPM, and that a steep concentration gradient of free DHA exists across the IPM such that the level of this fatty acid is about 8-fold higher i...