Cases of Rocky Mountain spotted fever (RMSF) in North Carolina have escalated markedly since 2000. In 2005, we identified a county in the Piedmont region with high case numbers of RMSF. We collected ticks and examined them for bacterial pathogens using molecular methods to determine if a novel tick vector or spotted fever group rickettsiae (SFGR) might be emerging. Amblyomma americanum, the lone star tick, comprised 99.6% of 6,502 specimens collected in suburban landscapes. In contrast, Dermacentor variabilis, the American dog tick, a principal vector of Rickettsia rickettsii, comprised < 1% of the ticks collected. Eleven of 25 lone star tick pools tested were infected with "Rickettsia amblyommii," an informally named SFGR. Sera from patients from the same county who were presumptively diagnosed by local physicians with a tick-borne illness were tested by an indirect immunofluorescence antibody (IFA) assay to confirm clinical diagnoses. Three of six patients classified as probable RMSF cases demonstrated a fourfold or greater rise in IgG class antibody titers between paired acute and convalescent sera to "R. amblyommii" antigens, but not to R. rickettsii antigens. White-tailed deer, Odocoileus virginianus, are preferred hosts of lone star ticks. Blood samples collected from hunter-killed deer from the same county were tested by IFA test for antibodies to Ehrlichia chaffeensis and "R. amblyommii." Twenty-eight (87%) of 32 deer were positive for antibodies to E. chaffeensis, but only 1 (3%) of the deer exhibited antibodies to "R. amblyommii," suggesting that deer are not the source of "R. amblyommii" infection for lone star ticks. We propose that some cases of rickettsiosis reported as RMSF may have been caused by "R. amblyommii" transmitted through the bite of A. americanum.