An enzyme-linked immunosorbent assay (ELISA) was used to quantitate interphotoreceptor retinoid-binding protein (IRBP) in tissues of monkey and other species using rabbit antiserum against monkey IRBP. A 1:7500 antiserum dilution was found optimal with the linear range extending to 250 micrograms IRBP/ml. The highest IRBP concentration was found in cannulation fluid of the monkey interphotoreceptor space, although vitreous and aqueous humors also contained IRBP. The presence of IRBP in the vitreous was confirmed by Western blot and 3H-retinol binding studies. The pineal gland of monkey and rat also was found to contain IRBP, as assessed by ELISA and immunocytochemistry; IRBP was below the limits of detection in turtle and chicken retina. IRBP levels were uniformly low in retinas of human cases with hereditary retinal degeneration, including retinitis pigmentosa (three cases) and choroideremia (two cases). The presence of IRBP in pineal as well as in the vitreous and aqueous humors may indicate a broader role for this putative retinoid-transport protein than previously suspected.