Bullous pemphigoid (BP) is an autoimmune blistering skin disease characterized in part by the presence of circulating and tissue-bound IgG antibodies directed against the epidermal basement membrane zone. IgG from over 95% of patients with BP have been shown to immunoprecipitate a 230-kD epidermal protein, BPAg1, which has been cloned and sequenced. Although sera from almost all patients with BP react with the 230-kD BP antigen the specific epitope(s) of BPAg1 that IgG binds is not known. We have generated fusion proteins from the 230-kD BP antigen cDNA and analyzed sera from patients with BP for binding to these fusion proteins by immunoblot. Sera from 21 of 30 (70%) patients with BP reacted with FP3A (amino acid 873-1193) compared to four of 13 (30%) normal subjects (p < 0.02). Sera from 10 of 30 (33%) patients reacted with FP7 (AA1623-1812) and to FP3 (AA1003-1193), compared to one of 22 (5%) and 0 of 19 (0%) controls, respectively. No significant reactivity was noted against two other fusion proteins (FP6, FP9). Twenty-four of 30 (80%) patients with BP reacted to at least one of three fusion proteins (FP3, FP3A, FP7) compared to three of 11 (27%) of the control subjects (p < 0.003). Fusion proteins FP3, FP3A, and FP7 are at the amino- or carboxyl-terminal regions of the putative central alpha-helical coiled-coil rod domain of BPAg1, which has been postulated to be involved in the self-aggregation of BPAg1. These findings demonstrate that patients with bullous pemphigoid react with multiple regions of BPAg1 and suggest that part of the pathologic consequences of these auto-antibodies in patients with bullous pemphigoid may be by the disruption of the normal self-aggregation of the BPAg1.