We have investigated expression of pemphigus vulgaris (PV) antigen, desmoglein 3 (Dsg3), and pemphigus foliaceus (PF) antigen, desmoglein 1 (Dsg1), in various tissues. Immunofluorescence studies suggested that Dsg1 and Dsg3 are preferentially expressed in the upper and lower epidermis, respectively. With immunoblotting of human epidermal extracts, all PV sera reacted with Dsg3 but not with Dsg1. By contrast, only half of PF sera reacted with Dsg1 but none reacted with Dsg3. These results confirm the distinct antibody specificity between PV and PF sera. They also suggest that PV sera contain antibodies against linear epitopes present even on the denatured antigen, while some PF sera contain antibodies only against conformational epitopes present on the native antigen. With immunoblotting of bovine desmosome preparations, certain PV sera and an anti-Dsg monoclonal antibody reacted with both Dsg1 and Dsg3. Affinity-purification of these PV antibodies suggested that the simultaneous reactivity with Dsg1 and Dsg3 was produced by two different subsets of antibodies and not by cross-reactivity of single antibodies. This study indicates that pemphigus serum is a useful probe with which we learn various aspects of keratinocyte biology.