Salicylate has been found to be an inhibitor of fatty acid synthesis in isolated rat hepatocytes. Half-maximal inhibition of fatty acid synthesis occurs at approximately 2 mM. The inhibitory effect of salicylate on fatty acid synthesis is not relieve by the addition of acetate, suggesting that salicylate inhibits the conversion of acetate into fatty acids. Acetyl-CoA carboxylase activity in homogenates of hepatocytes is not influenced by previous exposure of the intact cells to salicylate. Partially purified acetyl-CoA carboxylase, isolated and assayed in the absence of citrate, is markedly inhibited by salicylate. However, in the presence of 0.5 mM citrate, which is the concentration of this metabolite in the cytosol of the liver cell, salicylate activates the enzyme. Upon treatment of acetyl-CoA carboxylase with salicylate (in the absence or presence of citrate), followed by separation of enzyme and effector on a Sephadex G-25 column, the enzyme activity is enhanced as compared to the salicylate-free control, demonstrating that the inhibitory effect of salicylate (in the absence of citrate) is reversible, but not the stimulatory effect (in the presence of citrate). Salicylate inhibition of fatty acid synthesis by hepatocytes is not rapidly reversible; hepatocytes preincubated with salicylate followed by a wash procedure (centrifugation and resuspension) still show depressed rates of fatty acid synthesis from acetate upon further incubation. Salicylate was found to prevent pyruvate accumulation in hepatocyte suspensions observed in the absence of this compound; salicylate even induces the disappearance of pyruvate and lactate initially present in the cell suspension. This suggests that salicylate activates pyruvate and lactate consumption, which is most likely related to the well-known fact that salicylate uncouples oxidative phosphorylation. The latter action of the drug will stimulate citric acid-cycle activity. This causes an inhibition of fatty acid and cholesterol synthesis since acetyl units will be specifically channelled into the citric acid cycle and not into the lipogenic pathway. It is concluded that part of the inhibitory effect of salicylate on fatty acid biosynthesis is exerted at (a) step(s) in the conversion of acetate into fatty acids, acetyl-CoA carboxylase not being a target of this compound. In addition, salicylate prevents that pyruvate, generated by glycolysis, enters the lipogenic pathway. The latter effect of salicylate would also explain the observed inhibition of cholesterol synthesis by this compound.