In patients with pemphigus vulgaris, autoantlbodies against the desmosomal glycoprotein desmoglein 3 (Dsg3) cause blisters due to loss of keratinocyte cell-cell adhesion in the basal and immediate suprabasal layer of the deeper epidermis, leaving the superficial intact. Autoantibodies from these patients, usually bind to the cell surface of keratinocytes throughout the entire epidermis, as determined by indirect immunofluorescence. To explain this apparent paradox, we immunoadsorbed pemphigus vulgares sera with the extracellular domains of Dsg3 and desmoglein 1 (Dsgl) produced by insect cells infected with recombinant baculovirus. When adsorbed with extracellular domains of both Dsg3 and Dsg1, these sera no longer stained epidermis, demonstrating that most. if not all, of their cell surface reactivity can be attributed to antibodies against the extracellular domains of these desmogleins. Adsorption with only the Dsg1 extracellular domain left antibodies that stained only the basal and immediate suprabasal layers of the epidermis and immunoprecipitated only Dsg3, not Dsg1, from extracts of cultured cells synthesizing these molecules. In contrast, adsorption with only the Dsg3 extracellular domain Left antibodies that stained only the more superficial epidermis and immunoprecipitated only Dsg1. These data localize Dsg3 exactly to the area in the epidermis where Blisters occur in pemphigus vulgaris.