Though several vitamin deficiency frequently causes cutaneous lesions, the role of vitamins in the epidermal functions remains obsucure. Especially, the relationship between vitamin and stratum corneum lipid synthesis has not been well known. Stratum corneum lipids, particularly ceramides, are important components in epidermal permeability barrier. It has been reported that the level of ceramides is decreased in atopic dermatitis and aged skin. In this study, we examined the effects of vitamins on biosyntheses of sphingolipids including ceramides using cultured normal human keratinocytes. The rate of sphingolipids biosyntheses was measured by the incorporation of 1 4 C-serine into sphingolipids. When the cells were incubated with 1 to 10 μ M of niacinamide(NA) for 6 days, the synthesis rate of ceramide, cerebroside and sphingomyelin was increased dose-dependently up to 5-fold with 10 μ M NA compared with that of control. Other vitamins examined showed no effects. Furthermore, the activity of serine-palmitoyl transferase, a rate-limiting enzyme in sphingolipid synthesis, was increased in NA-treated cells. These results indicate that NA stimulates de novo synthesis of sphingolipids in human keratinocytes. Because the morphological changes of NA-treated cells were similar to those associated with the differentiation at a high Ca 2 + concentration, we examined the effect of NA on the differentiation of human keratinocytes at subconfluence. Both cornified envelope formation and expression of a differentiated type keratin K1, were accelerated by NA treatment at the same range of concentrations as above. Taken together, this study indicated that NA might improve epidermal permeability barrier by stimulating sphingolipid biosynthesis and/or by accelerating keratinocyte differetiation.