Acetyl-coenzyme A (CoA) is used in the cytosol of plant cells for the synthesis of a diverse set of phytochemicals including waxes, isoprenoids, stilbenes, and flavonoids. The source of cytosolic acetyl-CoA is unclear. We identified two Arabidopsis cDNAs that encode proteins similar to the amino and carboxy portions of human ATP-citrate lyase (ACL). Coexpression of these cDNAs in yeast (Saccharomyces cerevisiae) confers ACL activity, indicating that both the Arabidopsis genes are required for ACL activity. Arabidopsis ACL is a heteromeric enzyme composed of two distinct subunits, ACLA (45 kD) and ACLB (65 kD). The holoprotein has a molecular mass of 500 kD, which corresponds to a heterooctomer with an A 4 B 4 configuration. ACL activity and the ACLA and ACLB polypeptides are located in the cytosol, consistent with the lack of targeting peptides in the ACLA and ACLB sequences. In the Arabidopsis genome, three genes encode for the ACLA subunit (ACLA-1, At1g10670; ACLA-2, At1g60810; and ACLA-3, At1g09430), and two genes encode the ACLB subunit (ACLB-1, At3g06650 and ACLB-2, At5g49460). The ACLA and ACLB mRNAs accumulate in coordinated spatial and temporal patterns during plant development. This complex accumulation pattern is consistent with the predicted physiological needs for cytosolic acetyl-CoA, and is closely coordinated with the accumulation pattern of cytosolic acetyl-CoA carboxylase, an enzyme using cytosolic acetyl-CoA as a substrate. Taken together, these results indicate that ACL, encoded by the ACLA and ACLB genes of Arabidopsis, generates cytosolic acetyl-CoA. The heteromeric organization of this enzyme is common to green plants (including Chlorophyceae, Marchantimorpha, Bryopsida, Pinaceae, monocotyledons, and eudicots), species of fungi, Glaucophytes, Chlamydomonas, and prokaryotes. In contrast, all known animal ACL enzymes have a homomeric structure, indicating that a evolutionary fusion of the ACLA and ACLB genes probably occurred early in the evolutionary history of this kingdom.Acetyl-coenzyme A (CoA) is an intermediate metabolite that is juxtaposed between catabolic and anabolic processes. As the entry point for the tricarboxylic acid (TCA) cycle, acetyl-CoA can be considered the gateway in the oxidation of carbon derived from the catabolism of fatty acids, certain amino acids (e.g. Leu, Ile, Lys, and Trp), and carbohydrates. Furthermore, acetyl-CoA is the intermediate precursor for the biosynthesis of a wide variety of phytochemicals. Because membranes are impermeable to CoA derivatives, it can be inferred that acetyl-CoA is generated in at least four distinct metabolic pools representing the four subcellular compartments where acetyl-CoA metabolism occurs: plastids, mitochondria, peroxisomes, and the cytosol (Fig. 1). Therefore, plants should have distinct acetyl-CoA-generating systems in mitochondria (for the TCA cycle), in plastids (for de novo fatty acid biosynthesis), in peroxisomes (the product of -oxidation of fatty acids), and in the cytosol (for the biosynthesis of isoprenoids, flavo...