Borrelia burgdorferi isolates obtained from numerous locations and from different hosts in North Carolina, were compared to previously characterized strains of the Lyme disease spirochete and other Borrelia spp. The spirochete isolates were confirmed to be B. burgdorferi sensu stricto based on immunofluorescence (IFA) using a monoclonal antibody to outer surface protein A (Osp A [H5332]) and polymerase chain reaction (PCR) using a species-specific nested primer for a conserved region of the gene that encodes for flagellin. In addition, the isolates tested positive in Western blots with species-specific monoclonal antibodies for outer surface protein A and OspB (84c), and the genus-specific, monoclonal antibody to flagellin (H9724). Infectivity studies with several of these isolates were conducted using Mus musculus and Oryzomys palustris and the isolates exhibited markedly different levels of infectivity. This study demonstrates that B. burgdorferi sensu stricto is present and naturally transmitted on the Outer Banks and in the Coastal Plain and Piedmont regions of North Carolina.