Sera from patients with bullous pemphigoid (BP) contain autoantibodies that bind to the BP antigen, which is a component of the epithelial-stromal junction of the cornea. Previous studies, employing direct immunoelectron microscopy (IEM) on perilesional skin of patients have localized the BP antigen to the lamina lucida. On this basis, studies of corneal epithelial-stromal adhesion and wound healing have employed BP antigen as a marker of the lamina lucida of the corneal basement membrane zone (BMZ). The authors used indirect IEM with BP autoantibodies on frozen sections of cornea and found that the majority of the BP antigen is intracellular and is closely associated with the corneal epithelial hemidesmosome. Only a small amount of BP antigen appears to be extracellular, limited to the portion of the lamina lucida directly beneath individual hemidesmosomes. When rabbit corneal epithelium is extracted and analyzed by Western immunoblotting, BP autoantibodies recognize two polypeptides of molecular weights of 240 and 180 kilodaltons, which comigrate with BP antigens extracted from epidermis. BP autoantibodies are a specific marker of corneal epithelial hemidesmosomes and can be used as a probe to identify and study the role of hemidesmosomes in epithelial-stromal adhesion.