A cysteine proteinase was detected in extracts from cercariae of the trematode Diplostomum pseudospathaceum. The enzyme preferred protein substrates over synthetic, chromogenic peptides. The optimal pH for hydrolysis of substrates was 7.2 for azocoll, 6.4 and 7.6 for azocasein, 7.6 for azoalbumin, and 6.8 for N-benzoyl-L-arginine-4-nitroanilide. Elastin-Congo red and certain N-blocked L-aminoacyl- and L-peptidyl nitroanilides bearing L-phenylalanine, L-alanine, L-tyrosine, and L-leucine at the P1 subsite were not hydrolyzed. Thiol-reducing and divalent cation-complexing agents stimulated the proteinase activity, whereas thiol-blocking agents inhibited it. The relative molecular weight of the enzyme was approximately 40,000 as determined by SDS-PAGE. Detection of an identical proteinase in water after treatment of living cercariae with praziquantel suggests that the enzyme occupied the penetration glands in the larvae. Thus, when secreted by the parasite during invasion of an appropriate host, the enzyme might act as a penetration-promoting factor.