Lyme disease is prevalent over vast areas of land in the northern hemisphere. The etiologic agent is a spirochete, Borrelia burgdorferi, that is transmitted by Ixodes ticks. At least five species of anthropophilic ticks are competent vectors, and the causative agent or closely related species have been detected in 20 wild or domestic mammals and eight birds. The ability to isolate and culture B. burgdorferi from humans, ticks, and wildlife has significantly contributed to our understanding of the epizootiology of Lyme disease. Specific procedures for isolating borreliae from ticks and rodents in Barbour-Stoenner-Kelly medium are described. The authors have preserved in liquid nitrogen more than 500 isolates from ticks, wildlife, and a human.