The specific factors which induce blister formation in recessive dystrophic epidermolysis bullosa (RDEB) and epidermolysis bullosa simplex (EBS) were studied by culturing normal human skin with blister fluid from patients with RDEB and EBS. When skin from a healthy person was cultured with RDEB blister fluid, it developed a clean subepidermal blister with histology similar to that of a RDEB blister. The specific factor(s) which induced this subepidermal blister was inactivated by heat (60 degrees C, 30 min), trypsin digestion and by treating with EDTA, EGTA, alpha 2-macroglobulin, soybean trypsin inhibitor (SBTI) or N-ethylmaleimide (NEM), but was not affected by dialysis. These findings suggest that the active factor(s) in the blister fluid from patients with RDEB might include collagenase, neutral thiol protease and trypsin-like protease. By contrast, when normal skin was cultured with EBS blister fluid, this produced a clean intra-epidermal blister with histology similar to that of an EBS blister. The specific factor(s) inducing the intra-epidermal blister was inactivated by heat (60 degrees C, 30 min), trypsin digestion and by treating with NEM, but was not affected by dialysis, divalent cation chelators (EGTA, EDTA), alpha 2-macroglobulin, SBTI and pepstatin. These results suggest that the active factor(s) inducing the intra-epidermal blister in EBS might be a neutral SH-protease.