The hookworm Ancylostoma caninum induces human eosinophilic enteritis (EE), probably via allergic responses to its secretions. Cysteine and metallo-proteinases may be the components of these secretions that elicit hypersensitivity reactions. In order to characterize genes encoding cysteine proteinases (CP) secreted by A. caninum, an adult hookworm cDNA library was constructed and screened with a cloned fragment of a hookworm CP gene. This fragment was obtained using consensus oligonucleotide, CP-gene-specific primers in the polymerase chain reaction. cDNAs encoding two CPs were obtained from the library and sequenced. The first gene, AcCP-1, encoded a cathepsin B-like zymogen CP of 343 amino acids (aa), predicted to be processed in vivo into a mature CP of 255 aa. Closest nucleotide identities were to Haemonchus contortus cysteine protease (61%) and to human cathepsin B (60%). The second gene, AcCP-2, encoded a mature CP of 254 aa, that showed 86% identity to AcCP-1, and 58% and 47% identity to bovine cathepsin B and human cathepsin B, respectively. Rabbit antisera raised against recombinant AcCP-1 reacted with esophageal, amphidial and excretory glands in formalin-fixed, paraffin embedded sections of both male and female adult hookworms, and with an antigen of approx. 40 kDa in Western blot analysis of excretory/secretory products from adult hookworms. Together, these immuno-hybridization results strongly suggest that the CP encoded by the AcCP-1 gene is secreted by hookworms. These are the first reported CP genes from hookworms. Proteinases encoded by these genes may be responsible for the CP activity that we have shown previously to be secreted by adult A. caninum.