The zoonotic hookworm, Ancylostoma caninum, probably induces human eosinophilic enteritis by inducing allergic responses to its secretions. This species is already known to secrete metalloproteinases, but in other parasites, cysteine proteinases are involved in pathogenesis. We studied somatic extracts of A. caninum adults and infective larvae and adult excretory/secretory (ES) antigens for cysteine proteinase activity using fluorogenic peptide substrates and by gelatin and fluorogenic substrate polyacrylamide gel electrophoresis. Proteolytic activity was observed against the cathepsins L and B-specific substrate Z-phe-arg-AMC, against the plasmin substrate Boc-val-leu-lys-AMC, and against gelatin. The Z-phe-arg-AMC-hydrolyzing activity in ES antigens and in adult extracts was enhanced up to 15-fold by the reducing agent dithiothreitol (DTT), but was totally blocked by specific inhibitors of cysteine proteinases, including the peptidyl diazomethyl ketone Z-phe-ala-CHN2,E-64, leupeptin, and N-ethylmaleimide. In a similar fashion, gelatinolytic activity in ES antigens detected using substrate gels was enhanced by the addition of reducing agents and inhibited by Z-phe-ala-CHN2 and E-64. The DTT-enhanced, Z-phe-arg-AMC-hydrolyzing activity in ES antigens was active over a wide pH range (pH 5-9). Similar cysteine proteinase activity to that detected in ES antigens was present in extracts of adult and infective larvae of A. caninum. Because the substrate Z-phe-arg-AMC was specifically hydrolyzed, and because this hydrolysis was totally blocked by cysteine proteinase-specific inhibitors, ES antigens and tissue extracts of A. caninum clearly possess cysteine proteinase activity.(ABSTRACT TRUNCATED AT 250 WORDS)