Bat ticks, Carios kelleyi, from Iowa were examined for the presence of relapsing fever group borreliae. A novel spirochete was characterized by DNA sequence analysis of polymerase chain reaction amplicons for the 16S rRNA, flaB, and glpQ genes in either triturated tick pools or single ticks. All loci and the concatenated DNA sequence of 3,289 bases identified the Carios bacterium as a relapsing fever spirochete most closely related to, but distinct from, Borrelia turicatae. Spirochetes reactive with a Borrelia-specific monoclonal antibody were observed microscopically in the coxal fluid and salivary glands from one tick. These data confirm the presence of a novel species of relapsing fever spirochete in bat ticks and the potential for new enzootic foci for endemic relapsing fever that warrants further investigation. The name Borrelia johnsonii is proposed for this novel spirochete in honor of Dr. Russell C. Johnson.