Updated project metadata. Acetyl-CoA is a key metabolite in all organisms, implicated in transcriptional regulation, post-translational modification as well as fuelling the TCA cycle and the synthesis and elongation of fatty acids. The obligate intracellular parasite Toxoplasma gondii possesses two enzymes which produce acetyl-CoA in the cytosol and nucleus: the acetyl-CoA synthase (ACS) and the ATP-citrate lyase (ACL), while the branched chain α-ketoacid dehydrogenase (BCKDH) generates acetyl-CoA in the mitochondrion. Given the diverse functions of acetyl-CoA, we know little about the specific roles of distinct sub-cellular acetyl-CoA pools and how these impact overall parasite physiology. To assess the broad functions of nucleo-cytosolic as well as mitochondrial acetyl-CoA, we analysed the acetylome and total proteome of parasites lacking ACL/ACS or BCKDH.