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We examined the effect of repeated infestation of guinea pigs with Ixodes scapularis on the capacity of ticks to transmit Borrelia burgdorferi infection. Repeated challenges with nymphs or larvae lead to a reduction in duration of nymphal tick attachment and weight of recovered ticks consistent with the development of tick immunity. Only one of 18 I. scapularis-immune guinea pigs challenged with B. burgdorferi-infected nymphal ticks became infected, whereas 10 of 18 naive guinea pigs similarly challenged became infected. We conclude that tick immunity interferes with borrelial transmission.