The excreted/secreted proteinases of Haplometra cylindracea maintained in vitro, were found to hydrolyse the fluorogenic substrates, Z-ArgArg-NHMec and Z-PheArg-NHMec. This activity was shown to be typically that of cysteine proteinases, as turn-over of both substrates could be blocked by pre-incubation with peptidyl diazomethyl ketones. The biotinylated affinity reagent, biotin-Phe Ala-DMK, used in combination with Z-PheTyr(OBut)-DMK, was employed for the labelling and characterization of these cysteine proteinase activities. Three cathepsin B-like species were detected, with molecular weights of 48, 22–23 and 14 kDa, together with a cathepsin L-like enzyme, with a molecular weight of 55 kDa. The proteinases were also found to have hydrolytic activity towards the substrate, Z-GlyGlyArg-NHMec, which could be blocked by pre-incubation with either of the serine proteinase-selective reagents, Z-ArgP(OPh)2, or biotin-LysP(OPh)2, showing the activity to be trypsin-like. Using the biotinylated affinity label to characterize the trypsin-like enzymes revealed two molecular species with molecular weights of 20 and 24 kDa.