In vitro cultivation of Borrelia burgdorferi, the etiologic agent of Lyme spirochetosis, allows for the isolation and growth of this bacterium from infected tissues. However, continuous cultivation in modified Kelly medium causes a reduction in the number of detectable plasmids and the loss of infectivity in the white-footed mouse, Peromyscus leucopus. In an unpassaged culture of B. burgdorferi, nine plasmids were present, including seven linear plasmids ranging in size from 49 to 16 kilobases (kb) and two circular plasmids of 27 and 7.6 kb. The 7.6-kb circular and 22-kb linear plasmids were no longer detectable in spirochetes noninfective in white-footed mice, suggesting that a gene(s) encoding for factors responsible for infection may be present on one or more of these extrachromosomal elements. Furthermore, changes in spirochetal proteins and lipopolysaccharide-like material were observed also during early cultivation and may be related to loss of infectivity.