Acetyl-CoA is both an essential primary and secondary metabolite, however, the source of cytosolic acetyl-CoA has been unclear. We identified two non-overlapping groups of plant cDNAs that encode proteins similar to the amino and carboxy portions of human ATP-citrate lyase. In Arabidopsis, three genes encode the 45 kD ACLA subunit (ACLA-1, At1g10670; ACLA-2, At1g60810; ACLA-3, At1g09430) and two genes encode the 65 kD ACLB subunit (ACLB-1, At3g06650; ACLB-2, At5g49460). Co-expression of ACLA-1 and ACLB-2 cDNAs in Saccharomyces cerevisiae yields ATP-citrate lyase activity, indicating both polypeptides are required for activity. The Arabidopsis holoprotein has a molecular weight of 500 kDa, which corresponds to a heterooctomer with an A4B4 configuration. Consistent with the lack of targeting peptide sequence, subcellular fractionation studies show the ACL enzyme and the ACLA and ACLB subunits are located in the cytosol of the cell. ACLA and ACLB mRNAs accumulate coordinately in spatial and temporal patterns similar to those of mRNA for cytosolic acetyl-CoA carboxylase. Taken together, these results indicate that ATP-citrate lyase is encoded by the ACLA and ACLB genes of Arabidopsis and generates cytosolic acetyl-CoA. Antisense ACLA-1 Arabidopsis plants with only moderate (50%) reductions in ATP-citrate lyase activity have a complex dwarf or 'bonsai' phenotype. Anti-ACLA plants are proportionately reduced in size, possessing small organs comprised of small cells containing plastids with aberrant morphology, and fewer, often aberrant, seed. The roots are shorter, and highly branched; apical dominance is reduced; length of time to flowering and senescence is increased. Anti-ACLA plants have reduced cuticular wax and seed-coat flavonoids, but accumulate starch and vegetative anthocyanins. Accumulation of over ninety stress-related mRNAs is increased, while accumulation of sixteen photosynthetic-related mRNAs is reduced. Treatment with malonic acid, squalene, cholesterol, sitosterol or stigmasterol alleviates the anti-ACLA phenotype, while epibrassinolide or campesterol do not. Thus, a reduction in ACL activity places restrictions on the cytosolic acetyl-CoA pool and flow of available cytosolic acetyl-CoA from this pool is preferentially channeled to particular pathways. ACL is required for the survival of Arabidopsis and no other source of cytosolic acetyl-CoA can compensate.