In an attempt to characterize the enzyme(s) responsible for retinol esterification in hairless mouse epidermis, various subcellular fractions were incubated with [3H]retinol and the reaction products (retinyl esters) isolated by high-performance liquid chromatography. The microsomal fraction exhibited the highest esterifying activity and was stimulated by the addition of palmitoyl-CoA and dithiothreitol, but not by palmitic acid. Saturation kinetics with an apparent Km of about 6 microM for retinol were noted. Experiments with competitive and noncompetitive inhibitors of [3H]retinol esterification established that the epidermal enzyme was an acyl-CoA:retinol acyltransferase (ARAT; EC 2.3.1.76). The specificity for retinol was not absolute; a few closely related vitamin A alcohols were equally good substrates. The ARAT activity was not significantly altered by physiologic variations in the epidermal vitamin A content. In conclusion, mouse epidermis expresses ARAT activity which may be of importance for the regulation of vitamin A metabolism at the cellular level.