The pathogenic role of pemphigus autoantibodies and proteinases in epidermal acantholysis has been studied in organ cultures of normal human skin. Dose-dependent acantholysis occurred in skin explants cultured in medium containing 2-30 mg/ml of gamma-globulin from pemphigus serum. Acantholysis was not seen in explants cultured with 2 mg/ml pemphigus gamma-globulin although antibody binding to the epidermis was observed. Some degenerative changes in addition to acantholysis were present when 30 mg/ml pemphigus gamma-globulin was added to the medium. The addition of N,ethylmaleimide(NEM) and ethylene diamine tetraacetate(EDTA) prevented binding of pemphigus antibody to epidermis in culture. Soybean trypsin inhibitor and pepstatin had no effect on binding of pemphigus antibody to the epidermis but they did inhibit acantholysis in vitro. Our results suggest that pemphigus-induced acantholysis may be caused by at least 2 different types of enzyme.