For unknown reasons, the epidermal concentrations of retinol and its metabolite 3,4-didehydroretinol become characteristically changed during treatment with certain synthetic retinoids. In the present study we investigated [3H]retinol uptake and conversion to 3,4-[3H]didehydroretinol in cultured human skin exposed to six different retinoids. The tissue accumulation of [3H]retinol increased by 50–130% following addition of 1.3 μM of all-trans-retinoic acid, 13-cis-retinoic acid, or 3,4-didehydroretinoic acid (putative metabolites of retinol), and by 13–55% after addition of acitretin, arotenoid Ro 13-7410, or 4-(N-hydroxyphenyl)retinamide. The formation of 3,4-[3H]didehydroretinol was markedly inhibited (minus 80–90%) by the three putative retinol metabolites, moderately inhibited (minus 60–70%) by the aromatic retinoic acid analogues acitretin and arotinoid, and only slightly inhibited (minus 25%) by 4-(N-hydroxyphenyl)retinamide. Addition of citral and ketoconazole, substances known to interfere with the oxidative metabolism of vitamin A, diminished the formation of 3,4-didehydroretinol by 70 and 95%, respectively, but only marginally affected the retinol values. We conclude that the increase in retinol and marked reduction in 3,4-didehydroretinol concentrations in epidermis especially during treatment with 13-cis-retinoic acid are most likely due to diminished conversion of retinol to 3,4-didehydroretinol, but whether or not this reflects feedback inhibition of retinol metabolism or direct inhibition of a specific enzyme remains to be established. Nonetheless, in vitro analysis of 3,4-didehydroretinol biosynthesis appears to be a sensitive means for screening compounds for their ability to interfere with the cutaneous vitamin A metabolism.