The purpose of this study was to determine the ultrastructural localization of the epitopes recognized by two human monoclonal antibodies designated 5E and 10D, that were derived from virally transformed lymphocytes from a patient with bullous pemphigoid (BP). Previous immunoblotting showed that 5E but not 10D reacted with a 230-kDa protein in extracts of normal human skin. In the present study, indirect immunofluorescence showed that both 5E and 10D bound to the epidermal side of 1 M NaCl-separated normal human skin. Immunoperoxidase electron microscopy showed that 5E and 10D formed discontinuous focal deposits along the basal region of the epidermal basal cells in normal human skin in a similar localization to that seen with whole BP sera. Post-embedding immunogold electron microscopy demonstrated binding of both monoclonal antibodies to the intracellular components of hemidesmosomes. As far as we are aware, 5E and 10D are the first monoclonal antibodies to be described that recognize epitopes of BP antigen associated with hemidesmosomes and should serve as useful probes for future studies.