Recombinant human tissue inhibitor of metalloproteinase (rhTIMP) suppressed the ability of native human stromelysin to degrade [³H]transferrin in vitro. Maximum inhibition occurred at molar ratios (TIMP : stromelysin) of 2:1 and 1:1. Reduced and alkylated tissue inhibitor of metalloproteinases (TIMP) lost its ability to suppress stromelysin activity. rhTIMP also inhibited stromelysin from degrading proteoglycan monomer in vitro. When injected into the rat pleural cavity prior to stromelysin, rhTIMP inhibited the ability of the enzyme to degrade aggregating cartilage proteoglycan monomer. Marked inhibition of stromelysin-mediated proteoglycan degradation in vivo occurred at molar ratios (TIMP : enzyme) of 2:1 and 1:1, with less inhibition at molar ratios of 0.5:1 and 0.25:1. Reduction and alkylation prevented rhTIMP from suppressing stromelysin-mediated degradation of proteoglycan monomer in vivo. By comparison, an equimolar concentration of the serine proteinase inhibitor, α₁-proteinase inhibitor (α₁-PI), did not inhibit stromelysin activity in the rat pleural cavity. This study demonstrates that rhTIMP is effective in inhibiting native human stromelysin both in vitro and in vivo.