During terminal differentiation, mammalian epidermal lipids undergo striking changes in both composition and distribution. Phospholipids and neutral lipids are replaced by a mixture of ceramides and neutral lipids organized in intercellular lamellar bilayers. Whether all of these lipids and/or whether specific lipid classes regulate permeability barrier function is not known. When hairless mice were treated with acetone, the degree of barrier perturbation (measured as transepidermal water loss, TEWL) increased linearly with the amount of lipid removed. Moreover, virtually all lipid species appeared to be removed by acetone treatment. In contrast, the nonpolar organic solvent, petroleum ether, while removing greater amounts of lipids, provoked lesser barrier abnormalities. As determined by both quantitative thin-layer chromatography and histochemistry, petroleum ether selectively extracted nonpolar lipids leaving sphingolipids and free sterols in place. In petroleum ether-treated animals, subsequent acetone treatment removed additional sphingolipids and produced a dramatic increase in TEWL. A linear relationship existed for the quantities of sphingolipid removed and degree of barrier disruption in acetone-treated, but not petroleum ether-treated animals. These results support a relationship between the total lipid content of the stratum corneum and barrier function. Secondly, although the results demonstrate the participation of the total lipid mixture in the barrier, removal of nonpolar species alone appears to cause only a modest level of barrier disruption, while removal of sphingolipids and free sterols leads to a more profound level of barrier perturbation.