Acetate derived from ethanol oxidation is activated by cytosolic and mitochondrial acetyl-CoA synthetases before contributing to the extra-mitochondrial processes of fatty acid and 3-beta-hydroxysterol synthesis. Mitochondrially-generated acetyl-CoA is transferred to the cytosol via citrate and ATP-citrate lyase; this transfer is blocked by (-)-hydroxycitrate. Rats were injected IV with 3.3 mmol/kg of [2-3H,2-14C] acetate and IP with either 0.5 mmol/kg hydroxycitrate or saline. After one hour, the rats were killed and the incorporation of label was measured in liver fatty acids and 3-beta-hydroxysterols. The 3H/14C ratio was increased by 12 and 13% in the fatty acids and 3-beta-hydroxysterols of the hydroxycitrate-treated group. The lower ratio in the fatty acids and 3-beta-hydroxysterols derived from mitochondrially-generated acetyl-CoA is ascribed to a loss of 3H in the citrate synthase reaction. The data showed that (1) fatty acids and 3-beta-hydroxysterols syntheses use the same pool of cytosolic acetyl-CoA; and (2) in the absence of an isotope effect in the citrate synthase reaction, mitochondrially-generated acetyl-CoA contributes about 36% to lipogenesis from acetate.