Double subcutaneous vaccination of pups aged 3 and 4 months with 1,000 40-kr-irradiated Ancylostoma caninum larvae is more effective than double oral vaccination with the same preparation when resistance is measured by the establishment of adult hookworms resulting from an experimental challenge of normal larvae. In terms of resistance to the pathogenic effects of the challenge, as demonstrated by hematologic and coprologic findings and by clinical observation, both methods of vaccination are equally effective in protecting the vaccinates when compared with the severely affected challenge control dogs. The results suggest that the arrest of X-irradiated larvae in some somatic location on their migratory route following subcutaneous inoculation produces a better immunogenic stimulation of the host. Previous experiments have shown that treatment of the infective larvae of Ancylostoma caninum with X rays produces a characteristic attenuation (Miller, 1964). This attenuation was demonstrated by a reduction in the infectivity of the larvae, as measured by adult hookworm establishment in the intestine of the host, by a reduction in pathogenicity of the resultant burden, and by a sterilizing effect on the female worm at levels of larval irradiation of 40 kr or greater. A single subcutaneous infection with 1,000 40-kr-irradiated larvae was shown to confer a highly significant resistance to a subsequent challenge of normal larvae. Double subcutaneous vaccination with 40-krirradiated larvae is more than twice as effective as single vaccination (Miller, 1965a). A highly significant and satisfactory immunity can be produced in dogs of any age, even when the first dose of vaccine is administered as early as 72 hr after birth (Miller, 1965b). The present report describes experiments designed to investigate the immunogenic efficiency of a 40-kr-irradiated vaccine when the route of administration of vaccine and challenge larvae was varied. Received for publication 13 October 1964. MATERIALS AND METHODS These have been described elsewhere (Miller, 1964). This report comprises two separate experiments. The plan of experiment is illustrated in Table I. In each experiment, groups of pups without prior exposure to hookworm were vaccinated at 3 and at 4 months of age with 1,000 40-kr-irradiated A. caninum infective larvae, followed by a challenge of 1,000 normal infective larvae at the age of 5 months. At the time of challenge of the vaccinates, further groups of dogs aged 5 months, similar to the vaccinates in every respect except for the absence of prior exposure to hookworm, were infected with 1,000 normal larvae as challenge controls. On each occasion on which vaccine was prepared two further groups of dogs were infected, one group with irradiated larvae and the other with normal larvae. The necropsy worm burdens in these groups served as controls of normal larval infectivity and radiation attenuation for each batch of vaccine. Hematologic and fecal examinations were performed at suitable intervals throughout the experiments and the dogs were observed for clinical signs of ancylostomiasis.