Ceramides are the major component of the stratum corneum, accounting for 30%-40% of stratum corneum lipids by weight, and are composed of at least seven molecular groups (designated ceramides 1-7). Stratum corneum ceramides, together with cholesterol and fatty acids, form extracellular lamellae that are responsible for the epidermal permeability barrier. Previous studies indicated that beta-glucocerebrosidase- and sphingomyelinase-dependent ceramide production from glucosylceramides and sphingomyelins, respectively, is important for epidermal permeability barrier homeostasis. A recent study indicated that sphingomyelins are precursors of two stratum corneum ceramide molecular groups (ceramides 2 and 5). In this study, we have examined the role of glucosylceramides in the generation of each of the seven stratum corneum ceramide molecular groups. First, the structures of various glucosylceramide species in human epidermis were determined by gas chromatography-mass spectrometry, fast atom bombardment-mass spectrometry, and nuclear magnetic resonance. The results indicate that total epidermal glucosylceramides are composed of six distinct molecular groups, glucosylceramides 1-6. Glucosylceramide 1 contains sphingenine and nonhydroxy fatty acids, glucosylceramide 2, phytosphingosine and nonhydroxy fatty acids, glucosylceramide 3, phytosphingosine with one double bond and nonhydroxy fatty acids, glucosylceramide 4, sphingenine and alpha-hydroxy fatty acids, glucosylceramide 5, phytosphingosine and alpha-hydroxy fatty acids, and glucosylceramide 6, phytosphingosine with one double bond and alpha-hydroxy fatty acids. The nonhydroxy fatty acids typically have 16-24-carbon-length chains, whereas alpha-hydroxy fatty acids are limited to 24-, 25-, and 26-carbon chains. The sphingosine bases are C18 or C20 chains. Next, acylglucosylceramides and glucosylceramides were treated with beta-glucocerebrosidase and the ceramides released were compared with stratum corneum ceramides. Ceramide moieties of acylglucosylceramides and glucosylceramides 1, 2, 4-6 correspond to stratum corneum ceramides 1-7. These results, together with those of our previous reports characterizing epidermal sphingomyelins, indicate that all ceramide species, including omega-hydroxy fatty-acid-containing ceramides, are derived from glucosylceramides, and fractions of ceramides 2 and 5 are from sphingomyelins. Furthermore, structural analysis of glucosylceramides revealed that human epidermal glycosphingolipids display a unique lipid profile that is rich in very long chain hydroxylated (alpha- and omega-hydroxy) fatty acids and phytosphingosine.