Through yet unidentified mechanisms, squamous epithelial cells become committed to terminal differentiation after detachment from the basement membrane. In squamous cell carcinoma, these mechanisms seem to be disturbed. A murine monoclonal antibody, designated NCC-Lu-226 (IgG1, K), which recognizes an antigen expressed in basal cells of squamous epithelium at the epithelio-connective tissue border, was obtained. A cDNA clone encoding the antigen was isolated from a cDNA library by immunoselection. DNA sequencing and a database search revealed that this cDNA clone was identical to a hemidesmosomal transmembrane protein, bullous pemphigoid antigen 2 (BPA-2; also known as BPAG2, BP180, or type XVII collagen). Immunoelectron microscopy validated the specific reactivity of this monoclonal antibody with skin hemidesmosomes. Enhanced expression and abnormal distribution of BPA-2 was revealed immunohistochemically in various precancerous and cancerous tissues, including solar keratosis (4 of 5), Bowen's disease (3 of 5), invasive squamous cell carcinoma (7 of 7) of the skin, and squamous cell carcinoma of the lung (14 of 14), esophagus (12 of 13), and cervix (14 of 17). The specific expression of BPA-2 protein in squamous cell carcinoma was confirmed by RT-PCR and Northern hybridization. BPA-2 has possible phosphorylation sites and is actually phosphorylated in cultured keratinocytes and squamous cell carcinoma. The aberrant expression of BPA-2 may reflect dysfunction of the hemidesmosome that occurs as a relatively early event in multistep carcinogenesis of squamous epithelium.