Chronic ulcerative stomatitis (CUS) is a recently described mucocutaneous condition in which patients experience chronic, painful, ulcerative lesions of the oral mucosa. CUS is diagnosed by immunofluorescence studies that demonstrate antinuclear antibodies. These autoantibodies are specific for a protein, deltaNp63alpha, which is normally expressed in basal cell nuclei of stratified squamous epithelia. The purpose of this study was to characterize the autoimmune response in CUS. Protein antigens were produced by in vitro transcription/translation of polymerase chain-reaction (PCR)-amplified cDNAs. We used immunoblotting and immunoprecipitation experiments with serum from CUS patients to examine the (1) antibody isotype, (2) immunogenic functional domains of the deltaNp63alpha antigen, and (3) cross-reactivity with homologous p53, p73, and p63 proteins. Results demonstrate CUS patient antibodies to deltaNp63alpha, and 52% of cases have circulating IgA isotype antibodies. The N-terminal and DNA-binding domains are the immunodominant regions, and antibody cross-reactivity with p53, p63, and p73 isoforms is limited.