Serine protease inhibitors have important regulatory roles in angiogenesis, intravascular fibrinolysis, wound healing, and cell migration. In this study, the extracellular matrix secreted by cultured human keratinocytes, foreskin fibroblasts, and SV-40-transformed human skin fibroblasts was analyzed for serine protease inhibitors by substrate reverse zymography. We found that the extracellular matrix deposited by these cells contained three inhibitors (M(r) 33,000, 31,000, and 27,000). These inhibitors protected the degradation of gelatin by trypsin and elastase, and of casein by plasmin. In contrast, the gelatinolytic activities of thermolysin and papain were not inhibited. Compared to untreated cells, cells treated with phorbol 12-myristate 13-acetate showed a two- to 10-fold increase in the expression of these inhibitors. Cycloheximide and actinomycin D decreased the cellular expression of these inhibitors, suggesting the involvement of de novo protein and mRNA synthesis. Antitrypsin activity of these inhibitors was resistant to heat and sodium dodecylsulfate, but was lost after reduction of disulfide bonds. The inhibitors bound specifically to trypsin and could be eluted from a trypsin column in active form. Collectively, these data suggest that the extracellular matrix deposited by keratinocytes and dermal fibroblasts contains active serine protease inhibitors.