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We investigated if the monocytic Ehrlichia are totally dependent on their host cells for energy, or, as Rickettsia, are capable of some ATP synthesis in vitro. The Miyayama strain Ehrlichia sennetsu and the Maryland and Illinois strains of Enrlichia risticii were cultivated in a mouse macrophage cell line, separated from host cell constituents by procedures that included Renografin or Percoll gradient centrifugation, and tested after cryopreservation. Cells incubated without a metabolizing substrate contained little, if any, ATP. When the Ehrlichia cells were incubated for 1 hr at 34 C with glutamine, significant amounts of ATP were detected. The amounts of ATP attained with glutamine were decreased in some instances by the addition of atractyloside, an inhibitor of adenine nucleotide translocase in mitochondria, and were decreased consistently and to a greater extent by 2,4-dinitrophenol. When ATP, instead of glutamine, was added to the ehrlichiae, upon incubation the amount of ATP was markedly decreased. Comparable responses under all these conditions were obtained with Rickettsia typhi, although the final ATP levels were higher. Control preparations derived from uninfected mouse macrophages or from the discards of the Ehrlichia purification procedures contained negligible amounts of ATP, which were not increased by incubation with glutamine. We conclude that with respect to ATP metabolism, the monocytic Ehrlichia resemble Rickettsia more closely than Chlamydia, even though Ehrlichia resemble Chlamydia in their intracellular location in the phagosomes and in possibly having a developmental cycle. Reprints., Pub. in Proceedings of the National Academy of Sciences, v86 p1674-1678 Mar 1989.