Interphotoreceptor retinol-binding protein (IRBP) is a unique retinol carrier: it is a large glycoprotein existing only in the interphotoreceptor matrix (IPM), which is the extracellular material situated between and behind the photoreceptors of the neural retina. IRBP from bovine and human eyes carries endogenous retinol, as evidenced by the protein's fluorescence on gel-filtration (Sephacryl S-300) chromatography and on native (non-SDS) pore-gradient polyacrylamide gel electrophoresis. Bovine IRBP's retinol-binding sites are at most one-third occupied in light-adapted eyes and much less in dark-adapted eyes; this bleach dependence is partially reversible and suggests a role in vitamin A transport during the visual cycle. IRBP can be saturated with exogenous all-trans retinol; one ligand molecule binds per protein molecule. IRBP can be isolated rapidly from IPM by affinity adsorption onto con A-Sepharose; this preparation is 94% pure and yields 0.33 mg or 2.4 nmol of IRBP per bovine eye. An apparently homogeneous preparation can be obtained by subsequent passage through a Sepharose 4B column. IRBP is located only in the IPM, which is harvested by isotonic washing of the retina. (In the absence of this rinse to prepare IPM, the protein is found loosely associated with the retina, although not as a cellular or membrane component.) As the thoroughness of the retinal washing procedure is increased, the yield of IPM proteins (including IRBP) goes up; however, the same set of proteins, in the same ratios, always is retrieved, indicating that retinal-cell components do not contaminate seriously the IPM.