Confluent primary cultures of human retinal pigment epithelium (RPE) were incubated for 3-14 hr with 35S-methionine, 3H-leucine, or 2-3H-mannose, and the released proteins were analyzed by SDS-polyacrylamide gel electrophoresis. A 31,000-dalton protein constituted 10-70% of the released radioactive proteins. The peak was degraded by pronase and its synthesis was inhibited by cycloheximide. The incorporation of 2-3H-mannose into this protein, and its inhibition by tunicamycin, showed that the protein is glycosylated. Monensin (10(-5) M) also inhibited the release of the 31,000-dalton glycoprotein. The released 31,000-dalton glycoprotein from human RPE comigrated on gels with a protein present in extracts of human interphotoreceptor matrix, raising the possibility that the released protein corresponds to a component of the matrix.