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The study was conducted at sites of known transmission of Borrelia burgdorferi in east central Alabama. The objectives were to determine species of ticks present at these sites, their host associations, and species of ticks and small mammals naturally infected with B. burgdorferi. A total of 451 hosts were examined for ticks, including cotton mice, Peromyscus gossypinus (Le Conte); cotton rats, Sigmodon hispidus Say & Ord; southern short-tailed shrews, Blarina carolinensis (Bachman); house mice, Mus musculus L.; golden mice, Ochrotomys nuttalli (Harlan); and white-tailed deer, Odocoileus virginianus (Zimmermann). Ticks were examined for B. burgdorferi using indirect and direct fluorescent antibody assays. Ear biopsy samples from rodents were cultured in modified Kelly’s medium in attempts to isolate B. burgdorferi. A total of 859 Amblyomma americanum L., Dermacentor albipictus (Packard), D. variabilis (Say), Ixodes scapularis Say, and Rhipicephalus sanguineus (Latreille) were recovered from hosts and by dragging. A. americanum and I. scapularis accounted for 53.4% of all ticks collected. Nearly half of all ticks collected were examined for the agent. Spirochetes were detected in four nymphal and two adult A. americanum recovered from white-tailed deer and three larval I. scapularis recovered from cotton mice. No spirochetes were cultured from field-caught rodents.