Amblyomma americanum (L.) is a human-biting ixodid tick distributed throughout much of the southeastern United States. Rickettsia parkeri is a member of the spotted fever group rickettsiae and causes a febrile illness in humans commonly referred to as “Tidewater spotted fever” or “R. parkeri rickettsiosis.” Although the Gulf Coast tick, Amblyomma maculatum Koch, is the primary vector of R. parkeri, a small proportion of A. americanum have also been shown to harbor R. parkeri. The purpose of this investigation was to determine whether R. parkeri is spilling over into A. americanum in eastern Virginia and also to determine through laboratory experiments, whether A. americanum can acquire R. parkeri by cofeeding alongside infected ticks. Of 317 wild-caught, flat adult A. americanum tested from 29 counties and independent cities in coastal Virginia, a single female A. americanum was positive for R. parkeri, suggesting that R. parkeri is spilling over into this species, but at very low rates (<1.0%). Laboratory studies using guinea pigs indicated that nymphal A. americanum were able to acquire R. parkeri while feeding alongside infected A. maculatum and then transstadially maintain the infection. Nymphal A. americanum infected with Rickettsia amblyommii, however, were less likely to acquire R. parkeri, suggesting that infection with R. amblyommii may prevent R. parkeri from establishing infection in A. americanum .