A series of previous studies have indicated that the NC16a domain of the 180-kDa bullous pemphigoid antigen (BP180) is the most immunogenic and probably pathogenic region in both bullous pemphigoid and herpes gestationis. To confirm these previous results by a large-scale study, we examined serum from 154 bullous pemphigoid and 43 herpes gestationis patients using an immunoblot technique with the BP180 NC16a domain fusion protein, which had been prepared using cDNA obtained from a human keratinocyte cDNA library by polymerase chain reaction amplification. This fusion protein was recognized by 90% of bullous pemphigoid and 79% of herpes gestationis serum samples, but not by any other disease or normal control serum samples. These results indicate that this system is very sensitive and specific for the detection of anti-BP180 antibodies and should be useful for the diagnosis of various subepidermal bullous diseases.