Autoantibodies to type VII collagen are associated with the blistering diseases epidermolysis bullosa acquisita and bullous systemic lupus erythematosus. We showed previously that these autoantibodies recognize epitopes within the noncollagenous (NC1) region of type VII collagen. That region is composed of fibronectin type III homology units that may contribute to intermolecular cross-linking and basement membrane adhesion functions of type VII collagen. In this study, we defined the specific amino acid sequences recognized by these autoantibodies. By fusion protein analysis, sera from patients with epidermolysis bullosa acquisita and bullous lupus were found to react with two regions within the fourth (E-1) and eighth (E-2) fibronectin homology repeats, each consisting of approximately 100 amino acids, Affinity purification studies showed E-1 and E-2 to be independent and non-cross-reactive epitope regions. These regions were probed further by enzyme-linked immunosorbent assay analysis of overlapping octapeptide sets derived from the amino acid sequences of E-1 and E-2. The results showed two reactive, closely associated octapeptide sequences within each region, both lying in amphipathic portions of fibronectin type III homology repeats These studies identify short peptide sequences within the NC1 domain of type VII collagen that are targeted independently by autoantibodies. These sequences may play a direct role in determining the properties of type VII collagen that influence adhesion between this molecule and other basement membrane proteins, and their alteration by antibody binding may be the immunopathogenic event underlying epidermolysis bullosa acquisita and bullous lupus.