The concentration of 3,4-didehydroretinol, an epidermal metabolite of retinol, is changed both in association with keratinizing disorders and during treatment with certain synthetic retinoids. To delineate further the factors regulation biosynthesis of 3,4-didehy-droretinol, keratinocytes from three different layers of normal epidermis were prepared by trypsinization and incubated with [3H]-retinol for 20 h. Cell-bound [3H]-retinol and 3,4-[3H]-didehydroretinol were separated by high-performance liquid chromatography. The results indicate that the uptake of retinol and the synthesis of 3,4-didehydroretinol occur predominantly in undifferentiated cells from the basal and spinous layers of the epidermis.