Spirochetes were isolated from 71 subadult Ixodes dentatus removed from cottontail rabbits captured in Millbrook, N.Y., and in New York, N.Y. Spirochetes were also cultured from kidney tissues of six rabbits. While all isolates reacted with monoclonal antibody H9724, which identifies the spirochetes as borreliae, more than half did not bind with antibody H5332 and even fewer reacted with H3TS, both of which were produced to outer surface protein A of Borrelia burgdorferi. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein profiles of three isolates differed from one another and from all previously characterized B. burgdorferi strains from humans, ticks, and wildlife in North America. The 12 periplasmic flagella that originated subterminally from each pointed end of a rabbit Borellia isolate contrasted with the 11 or fewer flagella for B. burgdorferi reported previously from North America. Although DNA homology and restriction endonuclease analysis also revealed differences among a rabbit kidney isolate, an I. dentatus isolate, and B. burgdorferi B31, similarities were sufficient to lead us to conclude that the borreliae in rabbits and I. dentatus are B. burgdorferi. Enzyme-linked immunosorbent assay titers of sera from humans with diagnosed Lyme disease to rabbit tick B. burgdorferi were often similar to one another and to those recorded for a reference B. burgdorferi strain.