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Ixodes scapularis ticks acquired spirochetes while cofeeding with Borrelia burgdorferi-infected nymphs (donors) on uninfected naive gerbils. Overall, 19% (67 of 345) of the recipient nymphs randomly exposed to gerbils with donor nymphs acquired spirochetes by fluorescent antibody (FA) tests, 18% (62 of 345) by the polymerase chain reaction. In a second experiment, donor nymphs were placed on the left ears, recipient nymphs were placed on the left and right ears, and xenodiagnostic larvae were placed on the gerbils’ backs. Only recipient nymphs and larvae removed from the left ears were infected with B. burgdorferi. Infection rates were 47% (9 of 19), 87% (13 of 15) and 88% (14 of 16) in recipient nymphs placed on gerbils 0, 3, or 5 days after the donor nymphs, respectively, and 48% (12 of 25) in the larvae by FA analysis. Spirochetes appear to be acquired by cofeeding ticks from a localized infection near the feeding site rather than from a disseminated infection in the skin or blood.