The goal of the present study was to determine whether topically applied fatty acid esters enter into epidermal lipid metabolism. Of special interest with respect to epidermal function were the linoleate-rich O-acylsphingolipids thought to be essential in maintenance of the permeability barrier. Neonatal BALB/C mice were coated at 24 h intervals with 50 mg portions of methyl oleate or methyl palmitoleate. One day after the second application, the animals were sacrificed, and individual epidermal lipid classes were isolated. Fatty acid methyl esters were prepared from the isolated lipids and analyzed by gas-liquid chromatography. These analyses revealed that acylceramide was affected only slightly by application of either methyl ester. However, treatment with methyl oleate resulted in a significant replacement of linoleate by oleate in both acylglucosylceramide and phosphatidylethanolamine. This treatment also led to small increases in palmitoleate and possibly unresolved isomers of palmitoleate (16:1) in both lipids. Topical methyl palmitoleate resulted in a more complex pattern of substitution in both of these lipids. After methyl palmitoleate treatment, palmitate 16:0, 16:1 and vaccenate (18:1 delta 11) were elevated at the expense of linoleate in acylglucosylceramide. In the phosphatidylethanolamine, 16:1 and 18:1 delta 11 both increased while oleate (18:1 delta 9) and linoleate (18:2) decreased. These results demonstrate that topically applied fatty acid methyl esters can penetrate to the living cells of normal epidermis, enter into metabolism and significantly modify endogenous epidermal lipids.