Legionella pneumophila survives and replicates within a Legionella-containing vacuole (LCV) of amoebae and macrophages. Less is known about the carbon metabolism of the bacteria within the LCV. We have now analyzed the transfer and usage of amino acids from the natural host organism Acanthamoeba castellanii to Legionella pneumophila under in vivo (LCV) conditions. For this purpose, A. castellanii was 13C-labeled by incubation in buffer containing [U-(13)C(6)]glucose. Subsequently, these 13C-prelabeled amoebae were infected with L. pneumophila wild type or some mutants defective in putative key enzymes or regulators of carbon metabolism. 13C-Isotopologue compositions of amino acids from bacterial and amoebal proteins were then determined by mass spectrometry. In a comparative approach, the profiles documented the efficient uptake of Acanthamoeba amino acids into the LCV and further into L. pneumophila where they served as precursors for bacterial protein biosynthesis. More specifically, A. castellanii synthesized from exogenous [U-13C6]glucose unique isotopologue mixtures of several amino acids including Phe and Tyr, which were also observed in the same amino acids from LCV-grown L. pneumophila. Minor but significant differences were only detected in the isotopologue profiles of Ala, Asp, and Glu from the amoebal or bacterial protein fractions, respectively, indicating partial de novo synthesis of these amino acids by L. pneumophila. The similar isotopologue patterns in amino acids from L. pneumophila wild type and the mutants under study reflected the robustness of amino acid usage in the LCV of A. castellannii.