BACKGROUND Rickettsia amblyommii is a bacterium in the spotted fever group of organisms associated with the lone star tick (LST), Amblyomma americanum. The LST is the most commonly reported tick to parasitize humans in the southeastern US. Within this geographic region, there have been suspected cases of Rocky Mountain spotted fever (RMSF) where the causative agent, R. rickettsii, was not identified in the local tick population. In these areas, patients with clinical signs of RMSF had low or no detectable antibodies to R. rickettsii, resulting in an inability to confirm a diagnosis. METHODS R. amblyommii was cultivated from host-seeking LSTs trapped in Central Florida and propagated in ISE6 (Ixodes scapularis) and AAE2 (A. americanum) cells. Quantitative PCR targeting the 17-kD gene of Rickettsia spp. identified the genus of the organism in culture. Variable regions of groEL, gtlA and rompA genes were amplified and sequenced to confirm the species. The prevalence of R. amblyommii in LSTs within the geographic region was determined by qPCR followed by conventional PCR and direct sequencing. RESULTS Analyses of amplified sequences from the cultured organism were 100% homologous to R. amblyommii. The overall prevalence of Rickettsia spp. in the local population of LSTs was 57.1% and rompA sequence analysis identified only R. amblyommii in LSTs. CONCLUSIONS A Florida strain of R. amblyommii was successfully cultivated in two tick cell lines. Further evaluation of the new strain and comparisons to the other geographic strains is needed. The prevalence of this SFG organism in the tick population warrants further investigation into the organism's ability to cause clinical disease in mammalian species.