The cornified envelope of keratinocytes is an insoluble structure formed beneath the plasma membrane at the base of the stratum corneum. It is made by cross-linking precursor proteins by a membrane-associated transglutaminase. We have prepared monoclonal antibodies to the cornified envelope of cultured human keratinocytes and used these to identify precursor proteins using Western blotting. We have uncovered a number of precursors including involucrin and a 195 kD membrane-associated protein, which had previously been reported. Antibodies to these precursors, with the exception of the one to involucrin, reacted with the epidermis of other mammalian species, suggesting structural conservation in at least some envelope components.