Plasmodium falciparum asexual and gametocyte stages catabolize glucose and glutamine in a canonical tricarboxylic acid (TCA) cycle. Gametocytes, ring-stage parasite-infected, and uninfected red blood cells (RBCs) were suspended in medium containing either 13C-U-glucose or 13C-U-glutamine. (A) Metabolites were extracted from schizont-infected and uninfected RBCs (at 38 hours) and gametocytes (at 24 hours), and incorporation of 13C into polar metabolites was quantified by gas chromatography–mass spectrometry (GC-MS). Heat plots show enrichment (mol% containing one or more 13C carbons) after correction for natural abundance (n = 3 to 4). (B) Abundance of TCA-cycle isotopomers in schizont-infected RBCs. The x-axis indicates the number of 13C atoms in each metabolite (‘M’ indicates the monoisotopic mass containing no 13C atoms). Error bars indicate SD (n = 3 to 4). (C) Labeling of intermediates via the TCA cycle inferred from the isotopomer analysis. Grey boxes indicate fate of carbons in phosphoenolpyruvate (PEP) in indicated TCA-cycle intermediates. Both 13C2-acetyl-CoA and 13C3-oxaloacetic acid can be generated from 13C3-PEP, leading to formation of +2, +3, and +5 citrate. Uniformly labeled citrate can be generated through multiple rounds through the TCA cycle. Glutamine can enter the TCA cycle after its catabolism to α-ketoglutarate. Abbreviations: α-KG, α-ketoglutarate; Ac-CoA, acetyl-CoA; Asp, aspartate. Cit, citrate; Fum, fumarate; GABA, γ-aminobutyric acid; Glu, glutamate; Mal, malate; n.d., not detected; OAA, oxaloacetate; Pyr, pyruvate; Suc, succinate.