Borrelia burgdorferi is an etiologic agent of Lyme disease, the most common arthropod-borne disease in the United States (1,2). The bacterium, a member of the spirochete phylum, has a genome predominantly composed of linear DNA molecules (3,4). Formulating a medium in which B. burgdorferi grows in vitro was the first step toward a genetic understanding of the physiology and pathogenesis of the organism (5,6). The growth of B. burgdorferi as single colonies in solid medium (7–9) has facilitated mutant isolation by selection (10,11), although a defined medium for selection of auxotrophs is not currently available. The transformation system described in this chapter will be useful for manipulating the spirochete on a molecular genetic level.