Ambyomma americanum (L.) (Acari: Ixodidae) is an aggressive tick that feeds on humans during all postembryonic life stages. In many regions of the United States, it is the tick most commonly found attached to humans. Public health interest has grown recently, due to the recognition of new human pathogens transmitted by A. americanum and the expanding distribution of the tick. A. americanum is a vector of several bacteria pathogenic to humans. Ehrlichia chaffeensis and Ehrlichia ewingii cause moderate-to-severe febrile illness. “Rickettsia amblyommii,” a member of the spotted fever group Rickettsia, also has recently been implicated as a possible human pathogen based on serologic evidence from persons recovering from illness after a tick bite. We have determined the prevalence of infection of Ehrlichia chaffeensis, E. ewingii, “Borrelia lonestari,” and R. amblyommii within A. americanum ticks from 29 sites in nine states. Overall infection prevalences were 4.7% for E. chaffeensis (range, 0–27%), 3.5% for E. ewingii (range, 0–18.6%), 2.5% for B. lonestari (range, 0–12.2%), and 41.2% for R. amblyommii (range, 0–84.0%). In addition, 87 ticks (4.3%) were infected with two or more bacteria. This report documents new distribution records for E. ewingii, B. lonestari, and R. amblyommii and underscores the nonhomogeneous distribution of pathogen foci of infection. Additional surveillance throughout the range of A. americanum is warranted to increase physician and public awareness of the risk of disease to humans from exposure to the agents transmitted by this tick.