This study describes the presence of antibodies in sera from patients with cicatricial pemphigoid specific for a 168-kDa antigen expressed by buccal mucosa. Six cicatricial pemphigoid sera unreactive, with epidermal or dermal proteins in immunoblot assay were tested on mucosal protein extracts. Four of these sera labeled a mucosal 168-kDa antigen (M168) under reducing conditions. An additional cicatricial pemphigoid serum with circulating antibodies to 180-kDa bullous pemphigoid antigen (BPAg2) also labeled M168. None of these cicatricial pemphigoid sera reacted with the alpha, beta, or gamma subunits of laminin-5. Nitrocellulose elution studies showed that the M168 antigen is a basement membrane antigen and labeled the epidermal side of salt-split skin. Immunoaffinity-purified anti-M168 antibodies did not bind to the 230-kDa bullous pemphigoid antigen (BPAg1) or to the 180-kDa BPAg2. None of the control sera from healthy individuals or from bullous pemphigoid, pemphigus vulgaris, or pemphigus foliaceus patients reacted with Ml68. This study demonstrates the specificity of some cicatricial pemphigoid sera against a 168-kDa antigen that is different from the laminin-5 subunits and shares no epitopes with the antigens of bullous pemphigoid (BPAg1, BPAg2) or the epidermolysis bullosa acquisita.