Autoantibodies characteristic of autoimmune bullous skin diseases (AIBDs) can be detected by immunoblotting on epidermal, dermal, or bovine muzzle extracts. However, none of those substrates contain all the autoantigens involved in AIBDs, and the diagnosis requires the use of various substrates. Human keratinocytes were cultured under such conditions that they expressed the major autoantigens associated with AIBDs. Forty-two sera with antiepidermal antibodies were immunoblotted on the keratinocyte extract. Bands corresponding to desmoglein III, desmoglein I, BPAg2, BPAg1, and type VII collagen were found in 38 sera. Desmoplakins I and II were revealed by specific monoclonal antibodies. A review of the patients' charts showed a perfect correlation between the blots and the diagnoses of pemphigus vulgaris, pemphigus foliaceus, bullous pemphigoid, cicatricial pemphigoid, and epidermolysis bullosa acquisita. Four sera revealing no band typical of AIBD were from patients with no autoimmune skin disease. Therefore, a single extract of keratinocytes can be used for the differential diagnosis of AIBDs.