The Lyme disease spirochete, Borrelia burgdorferi Johnson, Schmid, Hyde, Steigerwalt & Brenner, was isolated from the blood of a dusky-footed wood rat, Neotoma fuscipes Baird, in the San Bernardino Mountains of southern California. Antigenic, protein, and molecular analyses demonstrated that the isolate varied slightly from most isolates of B. burgdorferi from northern California and was clearly distinct from other species of Borrelia that are endemic to the state. This is the first reported isolate of B. burgdorferi from southern California and demonstrates that the Lyme disease spirochete is enzootic in mountains near the major human population center of the state.