The retinal epithelium plays an important role in the storage and metabolism of retinoids in the eye. Studies were conducted to examine the enzymatic hydrolysis of retinyl esters by human retinal epithelial cells. Homogenates prepared from these cells were found to hydrolyze both the 11-cis- and all-trans-isomers of retinyl palmitate. Retinyl ester hydrolysis was time-, protein-, and pH-dependent. The 11-cis isomer was hydrolyzed at a rate which was approximately 20 times greater than that of the all-trans isomer. The 11-cis-retinyl palmitate hydrolase activity did not require detergents, unlike the all-trans-retinyl palmitate hydrolase activity, which required detergents for activity. The 11-cis-retinyl palmitate hydrolase activity was maximally active with the addition of 1.0% sodium taurocholate at about pH 8.5, was abolished by incubation at 50 degrees C for 10 min, and was quantitatively recovered in the pellet after centrifugation at 100,000 X g for 1 h. The rate of hydrolysis of 11-cis-retinyl palmitate became saturated with increasing concentrations of 11-cis-retinyl palmitate; under the assay conditions employed, the hydrolase activity had an apparent Km of 19 microM toward 11-cis-retinyl palmitate. All-trans-retinol and 11-cis-retinyl did not affect the rate of hydrolysis of 11-cis-retinyl palmitate, and addition of all-trans-retinyl palmitate only weakly inhibited the 11-cis-retinyl palmitate hydrolytic activities. These data indicate that the human retinal epithelium possesses distinct activities for the hydrolysis of 11-cis- and all-trans-retinyl esters and raise the possibility that these activities may provide a means of distinguishing the stereoisomers of retinol in this tissue.