The degradation of normal human skin by the human polymorphonuclear leukocyte proteinases cathepsin G and elastase, and by a human skin chymotrypsin-like proteinase that appears to be a mast cell constituent, was examined. Enzymes were incubated with fresh, split-thickness skin for up to 8 h; the tissue was examined ultrastructurally and immunohistochemically using antibodies to known basement membrane constituents. In all cases, the primary damage observed was at the epidermal-dermal junction. Elastase degraded the lamina densa leaving scattered and disorganized anchoring fibrils, dermal microfibril bundles, and normal-appearing collagen fibers. Immunohistochemically, type IV collagen, laminin, KF1 antigen, and EBA antigen were absent. The bullous pemphigoid antigen was present and localized on the basal cells. Epidermal-dermal separation produced by the chymotrypsin-like proteinases, cathepsin G, and the human skin proteinase, was confined to the lamina lucida. The lamina densa and sub-lamina densa fibrillar network remained intact. The human skin chymotrypsin-like proteinase produced extensive epidermal-dermal separation, while cathepsin G, at comparable concentrations, produced only focal separations. Immunohistochemically, all antigens were present after incubation with enzyme. The bullous pemphigoid antigen, however, was found on the epidermal side of the split, while laminin was found on the dermal side. These results show that the epidermal-dermal junction is highly susceptible to neutral serine proteinases located in mast cells and polymorphonuclear leukocytes. Although all the proteinases produce epidermal-dermal separation, the patterns and extent of degradation are different. The distinctive patterns of degradation may provide a clue to the involvement of these proteinases in skin diseases.