The expression of keratin proteins from meibomian glands and their correlation with skin epidermal keratins were determined. Keratin proteins were localized in both human and rabbit meibomian glands by indirect immunofluorescence using mouse monoclonal antibodies AE1, AE2 and AE3, which are known to react with human epidermal keratins as well as with keratins from other sources. Keratin proteins from rabbit meibomian glands were further isolated and characterized by SDS-PAGE and immunoblot using mouse monoclonal antibodies AE1 and AE3. Meibomian glands from human and rabbit showed similar immunofluorescent staining with each monoclonal antibody. AE1 antibody, which stains human basal epithelial cells of skin, stains all duct epithelial cells in the human but only the superficial duct epithelial cells in the rabbit meibomian gland. AE2 antibody, which stains human suprabasal epithelial cells of skin and is a marker for fully keratinized epithelia, stains the suprabasal epithelial cells of the central duct and ductules in both the human and rabbit meibomian gland. AE3 antibody, which stains all human epithelial cells of skin, stains all epithelial cells of the duct and ductules, as well as the basal epithelial cells of the acinus in both the human and rabbit meibomian gland. Keratins isolated from whole rabbit meibomian glands contained a 65-67 kD and 58 kD AE3-positive, and a 56.5 kD and 50 kD AE1-positive keratin protein. Expression of 65-67 kD/56.5 kD keratin proteins, and the immunofluorescent staining of the duct epithelium by the AE2 antibody, indicate that the meibomian gland duct epithelium is committed to the process of keratinization.