A heterogeneity of the bullous pemphigoid (BP) antigen initially described as a 220- to 240-kDa polypeptide doublet expressed in normal human skin was recently demonstrated. The aim of our study was to compare the heterogeneity of BP antigen by the immunoblotting technique in extracts of both human epidermis and keratinocyte cultures. Extracts of epidermal tissues obtained from plastic surgery skin samples and secondary keratinocyte cultures were analyzed for their immunologic content defined by 30 BP sera. Twenty-six out of 30 tested sera showed similar binding reactivities with one or several polypeptide bands in both extracts. These sera defined seven antigens of molecular weights ranging from 240 to 97 kDa. Three groups of sera reacting with three, two, or only one antigen could be distinguished in which 180, 200, and 220 kDa polypeptides represented the major BP antigens both in vivo and in vitro. The binding reactivity of most immunofluorescent negative sera demonstrated the sensitivity of the immunoblotting technique to evidence the heterogeneity of BP antigens and antibodies. Keratinocyte cultures represent a reproducible substrate for such analysis and offer a standardization attempt in comparative investigations in BP.