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The maintenance of Borrelia burgdorferi in a population of Peromyscus leucopus was investigated from 202 mark and recapture mice and 61 mice that were removed from a site in Baltimore County, Maryland. Borrelia burgdorferi infection was detected by culture and polymerase chain reaction (PCR) of ear tissue, and exposure to the spirochete was quantified by serology. Overall prevalence of B. burgdorferi, as determined by culture and PCR of ear tissue at first capture, was 25% in the longitudinal sample and 42% in the cross-sectional sample. Significantly more juvenile mice were captured in the longitudinal sample (18%) than in the cross-sectional sample (0%). Among 36 captured juvenile mice, only one was infected with B. burgdorferi; this contributed to a significant trend for infection with B. burgdorferi with age. Recovery from infection with B. burgdorferi was not detected among 77 mice followed for an average of 160 days. The incidence rate of infection with B. burgdorferi was 10 times greater in mice captured during two periods of high risk of exposure to nymphal Ixodes scapularis ticks compared with a period of low risk. Maintenance of B. burgdorferi in this population was dependent on indirect transmission of the organism from infected ticks to susceptible mice and development of chronic infection with the spirochete, which had no measurable effect on the survival of infected mice.