Vitamin A (vit A) metabolism was studied in freshly isolated and cultured human retinal pigment epithelial (RPE) cells obtained from postmortem donor eyes. Fluorometric determination of vit A in human RPE cells demonstrated that freshly isolated cells contained approximately 1.0 to 4.0 pg vit A/cell which decreased with increasing time in culture; after 48 hrs in culture cellular vit A was reduced 80%. High performance liquid chromatography (hplc) profiles of the retinyl esters in freshly isolated RPE cells showed the presence of 11-cis retinyl stearate and palmitate and all-trans retinyl stearate, palmitate and oleate; all-trans palmitate was the major ester. Hplc analyses of cell cultures supplemented with all-trans retinol, using fatty acid-free bovine serum albumin as a carrier, showed that the cells in primary and subcultures took up all-trans retinol and esterified it to form palmitate, stearate, and oleate. Palmitate was the major ester synthesized by the cells in primary cultures. In the subcultures the esters synthesized differed from that found in freshly isolated cells and in the cells in primary culture; in the subcultures, the overall synthesis of ester was reduced and oleate was more prominent. The esters that were synthesized in culture were all-trans; the formation of 11-cis isomers was not observed in human RPE cells in culture. Electron microscopy of retinol-supplemented cultures indicated that vit A doses up to 1.0 micrograms/ml had no obvious effects on the cells; at higher doses the cells no longer adhered to the culture surface.