We studied in vivo binding sites of pemphigus antibodies and the expression of pemphigus and desmosomal antigens by keratinocytes in various culture periods. Both pemphigus vulgaris (PV) and pemphigus foliaceus (PF) antibodies mainly bound to the desmosomal areas of the lesional skin. Desmoglein and PV antigens co-localized on the cultured normal human keratinocytes in both monolayers and stratified areas. PF antigens, frequently together with involucrin and suprabasal keratins, were expressed by stratified keratinocytes. The Western blotting study demonstrated two different desmogleins with molecular sizes of 130 and 150 kD. The 130-kD desmoglein bore PV antigenic epitopes. The 150-kD band increased in volume as cultured keratinocytes stratified. PV antigen expression on the 130-kD desmoglein precedes PF antigen formation occurring in keratinocyte differentiation.