Evidence for infection with the spirochete, Borrelia burgdorferi, was sought in Ixodes cookei and in groundhogs (Marmota monax) in southern Ontario, Canada, and ticks fed on experimentally inoculated hosts were examined for the spirochete. Borrelia burgdorferi was not detected by immunofluorescent examination of 110 larval, nymphal or adult I. cookei collected from the environment, or taken from humans and other animals. Three groundhogs inoculated with B. burgdorferi developed titers of 1:20 to 1:80 by the indirect immunofluorescent antibody test, but B. burgdorferi was not isolated from the spleens, kidneys, or urinary bladders of these animals. One of 30 wild groundhogs had an antibody titer of 1:20 to B. burgdorferi. Three (5%) of 59 I. cookei larvae fed on B. burgdorferi-infected hamsters became infected, in comparison with 23 (28%) of 82 I. dammini larvae fed on the same hosts. Borrelia burgdorferi was present in 5%, 16% and 4% of molted I. cookei nymphs fed on infected hamsters, rats or a groundhog, respectively; prevalences of infection in I. dammini fed on the same hosts were significantly (P < 0.05) higher (45%, 36%, and 23%, respectively), as was the intensity of infection. A naive groundhog on which I. cookei nymphs from an infected cohort fed did not become infected with B. burgdorferi, but it is uncertain whether an infected tick engorged on the experimental host. Ixodes cookei seems to be an inefficient vector of B. burgdorferi, and is unlikely to be significant in nature. Groundhogs are potential wildlife reservoirs of B. burgdorferi, based on their capacity to transmit infection to I. dammini.