Objective: In most organs there is a close link between metabolism and blood flow. Since AMPK has a key role in regulating tissue metabolism, we hypothesized that it may also control vascular tone. Therefore we studied the effects of AMPK activation on vasomotor reactivity of small blood vessels. Methods: Isolated, pressurized small mouse (C57BL/6J) mesenteric arteries (120-180mm) were studied in an organ bath (n=20). Smooth muscle cells were loaded with Fura 2-AM (10mM). The NOS and cyclooxygenase inhibitors L-NAME (30mM) and indomethacin (30mM) were used to inhibit the synthesis of endothelial NO and prostanoids, respectively. Vascular diameters and smooth muscle free calcium levels (Ca²⁺i) were continuously recorded. The vessels were preconstricted by elevating the extracellular potassium concentration to 60mM. All vasoactive compounds studied were applied to the organ bath. Results: The AMPK activator A769662 (A76) induced a rapid, dose dependent vasodilation which reached maximum at 300mM. This vasodilation was not associated with significant changes of calcium. In contrast, control vasodilations induced by the calcium entry blocker nifedipine went along with a significant reduction of Ca²⁺i below baseline values. Stepwise increase of the extracellular calcium concentration from 0-3 mM induced concomitant increases of Ca²⁺i and led to vasoconstriction of the vessels depolarised by potassium. A 76 did not alter the increases of Ca²⁺i but significantly reduced the accompanying vasoconstriction indicating a reduction of the sensitivity of the contractile apparatus to Ca²⁺i. Incubation of microvessels with the myosin-light-chain-phosphatase (MLCP) inhibitor Calyculin A (100nM) completely blocked the dilator effect of A76. Conclusion: AMPK activity potently reduces the smooth muscle tone of mouse microvessels. This is due to a reduction of the calcium sensitivity of the contractile apparatus which is most likely elicited by an activation of MLCP.