When added to the hepatocyte incubation medium, vanadate increased the rate of fatty acid synthesis de novo as well as the activity of acetyl-CoA carboxylase, whereas it had no effect on the activity of fatty acid synthase. On the other hand, and despite elevating the intracellular levels of malonyl-CoA, vanadate diverted exogenous fatty acids into the oxidation pathway at the expense of the esterification route. This was concomitant to an increase in carnitine palmitoyltransferase I activity. All these effects were not significantly different between periportal and perivenous hepatocytes and were also evident in cells incubated in Ca2(+)-free medium. Nevertheless, Ca2+ ions enhanced carnitine palmitoyltransferase I activity in isolated liver mitochondria. In addition, the effects of vanadate on acetyl-CoA carboxylase and carnitine palmitoyltransferase I were only evident in a permeabilized-cell assay, disappearing upon cell disruption and isolation of the corresponding cell subfraction for enzyme assay. Results show that vanadate exerts specific insulin-like and non-insulin-like effects on hepatic fatty acid metabolism, and suggest that the intracellular concentration of malonyl-CoA is not the only factor responsible for the regulation of the fatty-acid-oxidative process in the liver.