Epidermolysis bullosa simplex (EBS) is an uncommon genetic skin disease characterized by fragility of the basal keratinocytes and propensity to develop blisters. While a panel of antibodies against type IV collagen, laminin, and bullous pemphigoid antigen has been used to identify cases of EBS in frozen sections, we have found that a monoclonal antibody detecting cytokeratin in basal keratinocytes is useful in paraffin sections. Formalin-fixed, paraffin-embedded tissues from 12 patients with EBS were studied with the following monoclonal antibodies: MNF116 (DAKO, Carpinteria, CA), CAM 5.2 (Becton Dickinson, San Jose, CA), and AE1-AE3 mixture (Boehringer Mannheim Corp, Indianapolis, IN). Histologically, all the cases had focal vacuolization of the basal cell layer, with areas of dermal-epidermal separation. MNF116 was strongly positive in the basal keratinocytes, including the vacuolated ones, and demonstrated the presence of fragments of keratinocytes attached to the floor of the blister. CAM 5.2 stained sweat ducts only. AE1-AE3 was weakly positive in the basal cells, and almost completely negative on the fragmented basal cell keratinocytes. We consider that the immunostain with MNF116 in tissues fixed routinely in formalin and embedded in paraffin is helpful for the direct demonstration of the level of splitting in EBS.