The AMPK is an enzyme involved in the control of metabolism. Recent studies have shown that in parallel to its metabolic effects, AMPK can also affect blood flow in microvessels, thereby adding to the oxygen supply of the tissue. We have shown before that AMPK can induce short term vasodilation by reducing the cytosolic calcium by activation of SERCA1. Here we studied whether AMPK can also affect the calcium sensitivity of the smooth muscle contractile apparatus in these vessels. Studies were performed in segments of small murine mesenteric arteries which were freshly isolated. They were cannulated at both ends, loaded with FURA 2 AM to allow measurements of smooth muscle calcium changes and studied under constant pressure in an organ bath containing MOPS buffer. Diameter changes were recorded using videomicroscopy. Exposure of de-endothelialized vessels to the AMPK stimulator PT1 (30 μM) after blockade of SERCA (Thapsigargin 1µM) induced a slowly developing vasodilation of vessel pre-constricted with KCl (n= 5) which started after about 5 min and reached a maximum dilation of 53 +-5% after 20 min. Increasing the extracellular calcium concentration from 0 to 3 mM in several steps induced a stepwise constriction of vessels pretreated with high potassium solution to open Cav channels. When the vessels were pre-exposed to either of the AMPK activators, A769662, or PT1, the same increase in extracellular calcium (leading to similar increases of Ca++i) resulted in a significantly smaller calcium-dependent vasoconstriction as observed under control conditions (reduction by 17 %, n= 4 and 19%, n= 5 for A769662 and PT1, respectively, p<0.01). This inhibition of constriction went along with a siginificant increase of G-actin levels in vascular smooth muscle (n=4) . Moreover, microscopic analysis of the actin cytoskeleton in smooth muscle cells freshly isolated from mesenteric arteries (n= 12) yielded a reduction of actin branching points and a reduction of actin fibre thickness after treatemtn with PT1 as studied by Laser scanning microscopy using deconvolution techniques. These studies suggest that AMPK may reduce calcium sensitivity of the contractile apparatus by affecting cytoskeletal dynamics of microvascular smooth muscle. This mechanism could contribute the the long term maintenance of vasodilation without alteration of smooth muscle calcium levels which may be important for other non-contractile functions of vascular smooth muscle.