This is the first report of natural infection by Borrelia burgdorferi in the cotton rat Sigmodon hispidus. Nine B. burgdorferi isolates were obtained from ear tissues, urinary bladders, or both, by culturing tissues in BSKII medium. The rat from which the SI-3 isolate was cultured was from the same site (Sapelo Island, Georgia) as an infected cotton mouse Peromyscus gossypinus and Ixodes scapularis tick reported previously. The 8 B. burgdorferi isolates from rats in Florida included 1 (AI-1) from Amelia Island, 1 (FD-1) from Faver-Dykes State Park, and 6 (MI-3 through MI-8) from Merritt Island. The distance between Sapelo Island and Merritt Island is approximately 400 km. All B. burgdorferi isolates were characterized by indirect immunofluorescence using monoclonal antibodies to OspA (H3TS, H5332) and OspB (H5TS, H6831), polymerase chain reaction detection of specific B. burgdorferi B-31 DNA target sequences (ospA, fla, and a random chromosomal sequence), and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of spirochetal proteins. The phenotypic and genotypic characteristics of the isolates are discussed, as well as the probable importance of the cotton rat as a reservoir for B. burgdorferi in the southern United States.