The etiologic agent of Lyme disease, Borrelia burgdorferi Johnson, Schmid, Hyde, Steigerwalt & Brenner, was isolated repeatedly from dusky-footed wood rats, Neotoma fuscipes Baird, and California kangaroo rats, Dipodomys californicus Merriam, in northern California. All animals were collected in a region endemic for Lyme disease but for which the natural reservoir of B. burgdorferi was unknown. Similar attempts to isolate spirochetes from lizards, other species of rodents, jack rabbits, and deer between 1987 and 1991 were unsuccessful. Spirochetes isolated from wood rats and kangaroo rats were antigenically similar to strains of B. burgdorferi that had been isolated previously from the western black-legged tick, Ixodes pacificus Cooley & Kohls, in California. Similar enzootic cycles involving wood rats or kangaroo rats should be sought in other regions of the United States where the reservoirs of this spirochete are unknown.