The internal stratum corneum lipid composition was investigated in relation to depth in vivo in healthy human volunteers by extraction following one, three or five strippings. Automated multiple development high-performance thin-layer chromatography (AMD-HPTLC) and gas chromatography (GC) followed by normalized principal component analysis showed a decrease in the amount of lipids extracted after one, three and five strippings. Between levels 0, 1, 3 and 5 the stratum corneum lipid composition showed an increase in phospholipids and cholesterol-3-sulphate at level 3, a decrease in ceramide, cholesterol and free fatty acids after level 1, and a slight decrease in sterol esters at level 3. Lipids extracted after three strippings displayed a characteristic composition with an increase in the proportion of phospholipids and cholesterol-3-sulphate. Free fatty acid analysis in relation to depth revealed a decrease in the amounts of C14:0, C16:0, C16:1, C18:0 and C18:1 between levels 1 and 5 and an increase in the C24:0. A decrease in the unsaturated/saturated chain ratio with depth was also observed, reflecting a greater decrease in unsaturated than saturated free fatty acids. A decrease in the ratios of free fatty acids to cholesterol and free fatty acids to ceramides after three and five strippings, respectively, and previously reported results, confirm the importance of this level of stratum corneum lipids in skin barrier properties.