A 29 5 kDa cysteine proteinase was purified from medium m which mature Fasciola hepatica parasites were maintained. The N-terminal sequence (14 amino acid residues) of the purified protein is homologous to known cathepsin L proteinases, including a 27 kDa cathepsin L proteinase, also secreted by this parasite, which had been isolated previously in our laboratory (Smith e ta l , Mol Biochem Parasitol.,62,1-8,1993). The Nterminal sequences of the 29 5 and 27 kDa cathepsin L proteinases differ only in residue seven ( arginine and proline, respectively) Immunoblot studies, using antiserum that reacts with both cathepsin Ls, rule out the possibility of both enzymes ansing from a higher molecular sized parent molecule. The reaction kinetics of the two F hepatica cathepsin Ls on a variety of peptide substrates revealed that the two enzymes differ in their substrate specificity. Five peptide substrates that are cleaved with high affinity by the 29 5 kDa cathepsin L isolated m this study are not cleaved by the previously purified 27 kDa cathepsin L. The protein modifying reagent, tetramtromethane, affected the 29 5 kDa cathepsin L proteinase only, causing inactivation of the enzyme and changing its migration in polyacrylamide gels Our studies suggest that the two F hepatica cysteine proteinases represent two distinct subclasses within the cathepsin L class. The 29 5 kDa cathepsin L can cleave fibnnogen and produce a fibrin clot in vitro. This is the first demonstration of a cysteine proteinase with the ability to clot fibnnogen. The mechanism of clot formation of the cathepsin L was compared to bovine thrombin SDS-PAGE analysis of clots showed differences in enzyme activities between the two proteinases Cathepsin L was also not inhibited by the thrombin specific inhibitor hirudin Studies using peptides which prevent fibnn polymerisation showed that the fibrin clot formed by cathepsin L polymerises in a different manner to the clot formed by thrombin Clotting time assays in the presence of anti-polymerisation peptides and SDS-PAGE analysis of these clots showed that the cathepsin L fibrinogen cleavage site was located to the C-terminal of the thrombin cleavage site Therefore F hepatica cathepsin L forms a fibrin clot by a novel mechanism and may represent a means whereby F. hepatica may prevent excessive blood loss while migrating through host tissue.