The mammalian target of rapamycin (mTOR) is a conserved serine-threonine kinase that regulates cell growth and metabolism in response to nutrient signals. However, the specific involvement of mTOR in regulation of energy metabolism is poorly understood. To determine if signaling via mTOR might be directly involved in regulation of fatty acid metabolism in hepatocytes, we performed studies with rapamycin, a specific inhibitor of mTOR. Rapamycin-mediated inhibition of mTOR (18-48 hours) increased oxidation of exogenous fatty acids (46%-100%, respectively). In addition, esterification of exogenous fatty acids and de novo lipid synthesis were reduced (40%-60%, respectively). Consistent with inhibition of lipogenic pathways, rapamycin decreased expression of genes encoding acetyl-coenzyme A carboxylase I and mitochondrial glycerol phosphate acyltransferase. Non-insulin-dependent glucose transport and glycogen synthesis were decreased by 20% to 30%, whereas glucose utilization was unaffected by rapamycin. The data suggest that the hyperlipidemia observed with the drug in vivo is likely not the result of enhanced hepatic synthesis, but rather of delayed peripheral clearance. However, these results are consistent with the idea that mTOR may play a significant role, not only in "energy sensing," but also in regulation of energy production through profound effects on hepatic fatty acid metabolism.