ATP citrate (pro-S)-lyase (EC 4.1.3.8), a cytosolic enzyme that generates acetyl-CoA for cholesterol and fatty acid synthesis de novo, is a potential target for hypolipidaemic intervention. Here we describe the biological effects of the inhibition of ATP citrate-lyase on lipid metabolism in Hep G2 cells, and plasma lipids in rats and dogs, by using SB-204990, the cell-penetrant gamma-lactone prodrug of the potent ATP citrate-lyase inhibitor SB-201076 (Ki=1 microM). Consistent with an important role of ATP citrate-lyase in the supply of acetyl-CoA units for lipid synthesis de novo, SB-204990 inhibited cholesterol synthesis and fatty acid synthesis in Hep G2 cells (dose-related inhibition of up to 91% and 82% respectively) and rats (76% and 39% respectively). SB-204990, when administered orally to rats, was absorbed into the systemic circulation; pharmacologically relevant concentrations of SB-201076 were recovered in the liver. When administered in the diet (0.05-0. 25%, w/w) for 1 week, SB-204990 caused a dose-related decrease in plasma cholesterol (by up to 46%) and triglyceride levels (by up to 80%) in rats. This hypolipidaemic effect could be explained, at least in part, by a decrease (up to 48%) in hepatic very-low-density lipoprotein (VLDL) production as measured by the accumulation of VLDL in plasma after injection of Triton WR-1339. SB-204990 (25 mg/kg per day) also decreased plasma cholesterol levels (by up to 23%) and triglyceride levels (by up to 38%) in the dog, preferentially decreasing low-density lipoprotein compared with high-density lipoprotein cholesterol levels. Overall these results are consistent with the concept that ATP citrate-lyase is an important enzyme in controlling substrate supply for lipid synthesis de novo and a potential enzyme target for hypolipidaemic intervention.