Interphotoreceptor retinoid-binding protein (IRBP), a predominant protein in the interphotoreceptor matrix of the retina, has been implicated in transfer of retinoids between retinal pigment epithelium and photoreceptor cells. The interactions of IRBP with all-trans-retinol have been studied by three fluorescence-based methods and by measurements of binding of 3[H]-labeled all-trans-retinol to this protein. It was found that IRBP contains two sites with similar but not identical affinities for all-trans-retinol. The dissociation constant of the all-trans-retinol-IRBP complex at the first site was 0.1 microM, which is about 10-fold lower than previously reported values. The second site had about 2.5-fold lower affinity for all-trans-retinol as compared to the first site. Long-chain fatty acids were found in this study to displace all-trans-retinol from the stronger retinol-binding site on IRBP. Displacement of all-trans-retinol was used to study the interactions of fatty acids with this protein. It was found that docosahexaenoic acid (DHA C22:6n-3), an essential fatty acid which plays an important role in vision, had the highest apparent affinity for the site probed on IRBP of all the fatty acids studied.