Gelatinases produced by granulation tissue in culture were separated into two fractions when the conditioned medium was chromatographed on diethylaminoethyl-Sephacel; one contained latent and active gelatinases with molecular weight of about 74 kDa (low-Mr gelatinase), and the other contained an active gelatinase with molecular weight of about 112 kDa (high-Mr gelatinase) as estimated by gel filtration on Sephadex G-150. The former was unbound and the latter was bound to Zn-chelating Sepharose. Both low- and high-Mr gelatinases, however, occurred in mainly two bands corresponding to molecular weights of 64 and 57 kDa on sodium dodecyl sulfate-substrate polyacrylamide gel electrophoresis. Recombinant human tumor necrosis factor-alpha (TNF) markedly enhanced the production of gelatinases, especially high-Mr active gelatinase. Dexamethasone and hydrocortisone strongly suppressed TNF-mediated production of high-Mr active gelatinase, whereas indomethacin, piroxicam and mepacrine had no effect. The results suggest that steroidal anti-inflammatory drugs suppress a rapid collagen breakdown in granulation tissue through their inhibitory actions--one of which is the suppression of active gelatinase production enhanced by cytokines including TNF.