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The excretory-secretory product (ESP) derived from Cyathostominea in vitro was assessed, in terms of subunit composition, and proteolytic activity using as substrates azocasein and two synthetic fluorogenic peptides. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) resolved 13 subunits, and the presence of the protein cysteine proteinase activator dithiothreitol (DTT) revealed 21 subunits.DTTalso enhanced azocaseinolysis, and hydrolysis of carbobenzoxy-phenylalanyl-arginine-7-amido-4-methylcoumarin (Z-Phe-Arg-NHMec) and carbobenzoxy-arginyl-arginine-7-amido-4-methylcoumarin (Z-Arg-Arg-NHMec). At the optimum pH of 5.5, hydrolysis of Z-Phe-Arg-NHMec was three-fold greater than that of Z-Arg-Arg-NHMec suggesting that the proteolytic specificities of the ESP are more like those of papain or cathepsin L, rather than cathepsin B. In SDS-PAGE gelatin gels, DTT was a requirement for proteolysis by the ESP. Optimum resolution was at pH = 5.5, resolving six bands ranging from 114–20 kDa. Cysteine proteinase inhibitors abolished all gelatinolytic activity at the pH values tested. Such data indicate the presence of cysteine-class proteinases in the ESP of Cyathostominea.