Twenty patients with bullous pemphigoid were studied prospectively: sequential sera, in different phases of the disease, were collected over a period of approximately 2 years, the sera were tested using standard immunofluorescence techniques with salt-split and intact human tissue from different sites of the body (thigh, breast, oral mucosa, vagina); an early serum of each patient was tested by Western blotting. The concentration of circulating antibodies detected by the intact skin and intact mucous membranes was similar; split tissue was more sensitive than intact tissue. For eight of 19 patients, split vagina and occasionally split oral mucosa (in the same patients) were much less sensitive than all other tissues. Furthermore, there was a correlation between autoantibody reactivity with split mucous membrane tissues and clinical mucosal involvement. These results strongly suggest heterogeneity of antigens or epitopes expressed between tissues. In both split skin and mucosa all sera consistently detected an antigen on the epidermal side of the split regardless of the stage of the disease. Immunoblotting studies showed no correlation between specific antigens and mucosal expression or skin involvement.