In acne vulgaris, abnormal follicular keratinization is important for comedo formation, yet the precise mechanisms of comedogenesis are not known. The present study examined the interrelationship between sebum secretion rate (SSR), lipid content and water barrier function (WBF) of the stratum corneum (SC) in 36 acne patients and 29 control subjects. All major SC lipid classes were separated and quantified by thin-layer chromatography/photodensitometry. WBF was evaluated by measuring transepidermal water loss (TEWL), and the hygroscopic properties and waterholding capacity of the SC. The SSR over a period of 3 h was significantly higher in patients with moderate acne than in control subjects, but no significant difference was noticed between patients with mild acne and control subjects. Significant differences between patients with both moderate and mild acne and control subjects were noted in the amount of sphingolipids (ceramides and free sphingosine), but not for any other lipid classes. Furthermore in acne patients, lower amounts of sphingolipids were observed corresponding with a diminished WBF. These results suggest that an impaired WBF caused by decreased amounts of ceramides may be responsible for comedo formation, since barrier dysfunction is accompanied by hyperkeratosis of the follicular epithelium.