The AMPK is the only kinase that is thought to be able to regulate eNOS activity by phosphorylating it on more than one residue i.e. serine (Ser633 and Ser1177) and threonine (Thr495). However, as many of the reports on this topic were based solely on in vitro studies and showed very weak effects, the aim of this study was to determine the consequences of AMPK activation on eNOS phosphorylation and activity. In human umbilical vein endothelial cells a number of stimuli such as shear stress (12 dynes/cm2, 30 min to 4 hours), bradykinin (100 nM; 5 minutes) and VEGF (100 ng/mL, 30 min) elicited the activation of the AMPK and the phosphorylation of eNOS on Ser1177. As other kinases (Akt in the case of VEGF and Ca2+/calmodulin kinase in the case of bradykinin) are also able to phosphorylate eNOS we focused on the activation of the AMPK by different stimuli. Phenobarbital was found to be a rapid and potent activator of the AMPK in all of the endothelial cells tested and was without effect on the phosphorylation/activation of Akt or the CaMKb. Despite the pronounced activation of the AMPK there was no detectable change in the phosphorylation of the eNOS on Ser633 Ser1177 or Thr495. In endothelial cells from AMPKa1-/- and a2-/- mice, the fluid shear stress- induced phosphorylation of eNOS was identical to that observed in cells from wild-type littermates. Moreover, the NO-dependent relaxation to acetylcholine was comparable in aortae from wild-type and AMPKa-/- mice. VEGF (100 ng/mL) failed to elicit relaxation even in wild-type mice and the AMPK-activator rosiglitazone induced a pronounced NO- independent relaxation in the aortas from wild- type and AMPKa-/- mice. In the perfused mouse hindlimb, the AMPK activator bradykinin induced both NO-dependent and -independent relaxation, but neither was altered in AMPKa-/- animals. Taken together, we can find no evidence to indicate a major role of the AMPK in regulating eNOS phosphorylation or activity in isolated vessels or in cultured endothelial cells.