We have recently developed a novel immunoblotting system using bacterial fusion protein containing the NC16a domain, the most immunogenic region, of the 180 kD bullous pemphigoid (BP) antigen, which was shown to be highly sensitive and specific method to detect the antigen and to be very useful for the diagnosis of BP. In this study, using this immunoblot assay, we investigated sera obtained from 26 patients who showed atypical clinical features. The sera of these cases showed in general very weak reactivity with the basement membrane zone on immunofluorescence, and very weak and variable results in detection of either the 230 kD or 180 kD BP antigen with immunoblotting of human epidermal extracts. However, with immunoblotting of the BP180 NC16a domain fusion protein, this protein was detected by 21 out of the 26 cases. These results indicated that this new assay system is the most sensitive method to detect BP antigen, and should be very useful for the diagnosis for these atypical cases.