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Various routes of Borrelia burgdorferi infection were studied in laboratory rats. Three-week-old Lewis rats were inoculated either intradermally (i.d.), intraperitoneally (i.p.), or oronasally (o.n.) with serial 10-fold dilutions of B. burgdorferi. Thirty days later, groups of rats were killed and serology, splenic culture, and histology were used to evaluate infection. Rats were successfully infected i.d. with 102–4 organisms or i.p. with 104–5 organisms. Neither three-day-old nor three-week-old rats were successfully infected o.n. with up to 106 organisms. For contact transmission, three-day-old or three-week-old inoculated rats were housed with unexposed littermates for 30 days. Inoculated rats became infected but contact rats remained free of infection. To study in utero transmission, five pregnant female Lewis rats were inoculated i.p. with 106 spirochetes at four days gestation. Although adult females seroconverted or had positive splenic cultures at 20 days gestation, the placentas and fetuses were uniformly culture-negative. Venereal transmission from seven infected females or six infected males to uninfected rats of the opposite sex was not demonstrated.