Infection with Ancylostoma caninum, an intestinal hookworm of dogs, can cause debilitating and potentially life-threatening disease. In the current study, protective immunity to hookworm infection was induced in dogs following vaccination with irradiation-attenuated third-stage larvae (L3) with significant reductions in both worm (P<0.03) and faecal egg counts (P<0.0004) following a challenge infection. Vaccination with irradiated L3 and challenge with infective L3 stimulated a dominant antibody response to antigens of less than 20 kDa in an excretory/secretory extract from adult parasites. Immunoscreening of an adult A. caninum cDNA library with antisera from the vaccine trial identified a number of clones. The three clones with the strongest immunoreactivity proved to be identical and encoded a peptide with similarity to the N-terminal domain of the tissue matrix metalloproteinase inhibitor (TIMP)-2 mammalian tissue metalloproteinase inhibitor family.