Vitamin A is stored in cells as long-chain fatty acyl esters of retinol. Esterification in many tissues is catalyzed in part by acyl-CoA:retinol acyltransferase (ARAT). Since the lacrimal gland contains stores of retinyl esters, it was the goal of this study to determine whether the lacrimal gland contains ARAT activity. Rabbit lacrimal gland microsomes incubated with 3H-retinol synthesized retinyl esters. The reaction rate was stimulated 30-fold in the presence of a fatty acyl-CoA generating system, producing a mixture of esters including retinyl laurate, retinyl linoleate, retinyl palmitate, and retinyl stearate as determined by reverse-phase HPLC. Retinyl palmitate was synthesized at 1944 pmole/mg protein/30 min, representing 50% of total ester synthesis, and this activity was directly proportional to microsomal protein concentration. In the presence of 180 microM 3H-retinol and 100 microM palmitoyl-CoA, retinyl palmitate was synthesized at 175-220 pmole/mg/min, and the reaction fit Michaelis-Menten kinetics as a function of retinal concentration (theoretical Vmax = 329.4 pmole/mg/min). Lauroyl CoA and stearoyl CoA, but not linoleoyl CoA, were as effective as palmitoyl CoA as substrates for the reaction. The enzyme activity was inhibited by p-chloromercuriphenyl sulfonic acid and Na-taurocholate. The data show that the lacrimal gland synthesizes retinyl esters and that the characteristics of synthesis are consistent with the presence of acyl-CoA:retinol acyltransferase in lacrimal gland.