To determine whether cottontail rabbits (Sylvilagus floridanus) maintain an enzootic cycle of transmission of the Lyme disease spirochete (Borrelia burgdorferi), we examined the prevalence of infection in ticks and rabbits in a location in which rabbits were abundant. Of 72 unfed nymphal Ixodes dentatus swept from vegetation, 32% were infected by this spirochete, as determined by darkfield microscopy and indirect immunofluorescence using monoclonal antibody H5332. Infected ticks were reared from larvae feeding on each of 11 rabbits taken from the same site. Of 50 rabbits sampled there over a period of 2 years, sera of > 90% reacted with B. burgdorferi antigen by ELISA and by immunoblotting. Deer ticks (I. dammini) comprised <10% of ticks found on rabbits. We conclude that rabbits perpetuate the agent of Lyme disease in an enzootic cycle where rabbit-feeding Ixodes are abundant, that intensity of transmission is independent of the zoonotic cycle in mice, but that infection may occasionally be exchanged between these cycles.