The identity of two differentiation markers of human epidermis, filaggrin and a Concanavalin A (Con-A) reactive glycoprotein of 37 kD, has been studied. Human epidermis was extracted in Nonidet P-40 buffer, and the soluble proteins were separated by two-dimensional electrophoresis. Con-A reactive glycoproteins were identified by incubating gels with iodinated lectin followed by autoradiography. Identical, parallel gels were electrophoretically transferred to nitrocellulose paper and filaggrin-related molecules labeled by the specific monoclonal antibody AKH1. We found that the 37-kD Con-A reactive component was resolved by two-dimensional gel electrophoresis into several glycoproteins and that the lectin Con-A does not bind to filaggrin. Under these conditions, the anti-GP37 serum failed to identify any component. However, when applied to human keratinocyte culture extract, AKH1 and the anti-GP37 serum reacted in a similar way. These data show 1) that the 37-kD band is not homogeneous but contains distinct markers of differentiation (filaggrin and Con-A reactive glycoproteins) and 2) that the GP37 antibody's specificity is for the filaggrin precursor.