Epidermolysis bullosa acquisita is a subepidermal blistering disease in which patients have autoantibodies against the non-collagenous domain of type VII collagen. Starting with previously isolated 1-kilobase pair (Kb) cDNA for this au- toantigen, we isolated overlapping cDNAs with a combined open reading frame of -&∼3.2 Kb, encoding most (&∼115 kilo- daltons [KDa]) of the N-terminal non-collagenous domain of type VII collagen. To localize immunogenic domains, we produced maltose-binding fusion proteins with cDNA encoding different portions of this autoantigen. These cDNA fragments scan from 5' to 3' of this non-collagenous domain and overlap each other. An immunoblot analysis of these fusion proteins with eight epidermolysis bullosa acquisita patient sera demonstrated that each patient serum binds to different regions of this molecule and that epitopes for these patient sera locate throughout this autoantigen. These data suggest that multiple epitopes on the N-terminal non-collagenous domain of type VII collagen are recognized by circulating autoantibodies in patients with epidermolysis bullosa acquisita.