Clostridium histolyticum supernatant, thanks to intrinsic proteases, exerts cytotoxic effect on cells and tissues. In our study we compared the ability of five different protease inhibitors to block cytotoxic activity of C histolyticum supertanant: aprotinin, leupeptin, phenylmethylsulphonyl fluoride (PMSF), L-1-chloro-3-[4-tosylamido]-7-amino-2-heptanone-HCl (TLCK) and chymostatin. Inhibitors of serine proteases nearly completely blocked cytotoxic effect, especially in lower supernatant concentrations. At the same time TLCK, known to irreversibly block clostripain obtained from C histolyticium, blocked enzymatic activity of clostripain, but not cytotoxic effect of the supernatant. We conclude that the remaining cytotoxicity must be the effect of the activity of another factor that is contained in the supernatant and that can be isolated and tested in the presence of TLCK. (c) 2005 Elsevier Inc. All rights reserved.