In the vertebrate retina, the retinal pigment epithelium (RPE) performs specific functions critical to the normal process of vision. Although some of these functions are well documented, molecular data are still scarce. Using the RPE-specific monoclonal antibody RPE9, raised against human RPE cells, we have identified a novel 65 kD protein, conserved in mammals, birds, and frogs. This RPE-specific protein was found to be nonglycosylated. It was most effectively solubilized in the presence of detergent suggesting that it is associated with the RPE cell membranes. Its partitioning in the detergent phase of Triton X-114 and its solubilization in 0.75 M and 1.0 M KCl suggest that it interacts with the membrane either through a polypeptide anchor or charged amino acids. Cell fractionation by differential solubilization and differential centrifugation demonstrated that the protein was preferentially associated with the microsomal membrane fraction, where it is the major protein. Developmental expression of this 65 kD protein was examined in neonatal rats. Morphologically well-differentiated RPE cells did not express the 65 kD protein at birth. However, expression was detectable at postnatal day 4, that is, one to two days before the photoreceptors develop their outer segments, suggesting that the expression of the 65 kD protein may be coordinated with other developmental events in the intact retina. This is further supported by the fact that RPE cells in confluent culture lose the expression of this protein within two weeks, while they maintain their characteristic epithelial morphology. Because of its specificity, its evolutionary conservation, and its timing of expression, it is possible that this protein may be involved in one of the key roles of RPE and as such is an important molecular marker for RPE differentiation.