Abstract The proteolytic activity of worm homogenates prepared from the third stage larval (L3) and adult stages of the ovine gastro-intestinal nematodes, Nematodirus battus, Ostertagia circumcincta, Trichostrongylus colubriformis, Trichostrongylus vitrinus and Haemonchus contortus, and the rodent intestinal nematode, Nippostrongylus brasiliensis, has been examined using the protein substrates azocasein, azocoll and elastin-orcein. Activity detected in third stage larvae was usually higher than that observed in the adult. Species and stage differences were demonstrated. The in vitro release of proteolytic activity, detected using protein substrates and specific low molecular weight peptide substrates, was, similarly, shown to exhibit a degree of species and stage specificity. Acetylcholinesterase and lactate dehydrogenase activities were determined as reference ‘secretory’ and ‘non-secretory’ enzymes, respectively. Three separate peaks of ‘tryptic’ activity were detected following anion-exchange chromatography of culture fluids derived from adult Ostertagia circumcincta. These peaks could be ascribed to different proteinase classes on the basis of inhibitor sensitivity.