It has long been assumed that Ehrlichia chaffeensis (Anderson, Dawson & Wilson), is not transmitted transovarially in the lone star tick vector Amblyomma americanum (L.). To test this hypothesis, three beagle dogs, Canis familiaris (L.) (Carnivora: Canidae), were subcutaneously infected with E. chaffeensis (Arkansas strain). Uninfected nymphal lone star ticks were placed on the infected dogs and allowed to feed to repletion. These nymphal ticks were allowed to molt, and five of five adult female ticks sampled were confirmed to be infected by polymerase chain reaction (PCR). Forty infected adult ticks, the majority of which were female with several males included to stimulate feeding, were then placed on two uninfected dogs. Fourteen females were removed early and the rest were allowed to feed to repletion. After feeding to repletion, the six remaining females detached and two of these females were tested preoviposition, whereas the remaining four were allowed to lay eggs. All six of these fully engorged females tested negative by PCR. The egg clutches laid by four engorged adult females, and the larvae that hatched from these eggs along with larvae from a previously untested egg clutch, were shown to be uninfected by PCR. This seems to support the long-held assumption of lack of transovarial transmission. Also of interest, we found that these PCR-confirmed infected adult ticks reared in the laboratory did not transmit E. chaffeensis to uninfected dogs.