Itis generally accepted that endothelial cells generate most of their ATP byanaerobic glycolysis and that very little ATP is derived from the oxidation offatty acids or glucose. Previously, we have reported that, in cultured humanumbilical vein endothelial cells (HUVECs), activation of AMP-activated proteinkinase (AMPK) by the cell-permeable activator 5-aminoimidazole-4-carboximideriboside (AICAR) is associated with an increase in the oxidation of3H-palmitate. In the present study, experimentscarried out with cultured HUVECs revealed the following: (1) AICAR-inducedincreases in palmitate oxidation during a 2-hour incubation are associatedwith a decrease in the concentration of malonyl coenzyme A (CoA) (an inhibitorof carnitine palmitoyl transferase 1), which temporally parallels the increasein AMPK activity and a decrease in the activity of acetyl CoA carboxylase(ACC). (2) AICAR does not stimulate either palmitate oxidation when carnitineis omitted from the medium or oxidation of the medium-chain fatty acidoctanoate. (3) When intracellular lipid pools are prelabeled with3H-palmitate, the measured rate of palmitateoxidation is 3-fold higher, and in the presence of AICAR, it accounts fornearly 40% of calculated ATP generation. (4) Incubation of HUVECs in aglucose-free medium for 2 hours causes the same changes in AMPK, ACC, malonylCoA, and palmitate oxidation as does AICAR. (5) Under all conditions studied,the contribution of glucose oxidation to ATP production is minimal. Theresults indicate that the AMPK-ACC-malonyl CoA-carnitine palmitoyl transferase1 mechanism plays a key role in the physiological regulation of fatty acidoxidation in HUVECs. They also indicate that HUVECs oxidize fatty acids fromboth intracellular and extracellular sources, and that when this is taken intoaccount, fatty acids can be a major substrate for ATP generation. Finally,they suggest that AMPK is likely to be a major factor in modulating theresponse of the endothelium to stresses that alter its energystate.