The cornified outer cells of mammalian epidermis possess a monolayer of omega-hydroxyceramides that are ester-linked to the exterior of a cross-linked protein envelope. In the present study, conclusive evidence was sought on which of the ceramide hydroxyl groups are involved in the linkage to protein. This was obtained by derivatizing all free hydroxyl groups in isolated solvent-extracted porcine stratum corneum using triisopropylsilyl (TIPS) chloride in pyridine in the presence of silver nitrate. After an 18-h reaction, the tissue was recovered, rinsed, and the derivatized ceramides were then released from protein linkage by hydrolysis with 1M KOH in 95% methanol. This gave a single ceramide product that was shown by nuclear magnetic resonance to contain two triisopropyl groups. Acetylation of the product using acetic anhydride in pyridine resulted in a downfield shift of the NMR signal for the omega-methylene protons, showing that it was the omega-hydroxyl that was free in the initial reaction product, and subsequently was acetylated. These results show that all of the omega-hydroxyceramides of corneocyte lipid envelopes are attached to protein through their omega-hydroxyl groups.