Endothelial cells are key regulators of cardiovascular homeostasis, including the maintenance of blood flow and vascular tone. Endothelium-derived nitric oxide (NO) promotes vasodilatation and inhibits platelet aggregation, leukocyte adherence and VSMC proliferation, which all have a profound influence on blood flow, vascular remodelling and angiogenesis. Endothelial dysfunction, tissue injury and inflammation are associated with activation of the vascular endothelium and increased adhesion of circulating leukocytes. These are critical early events in the development of atherosclerosis. Endothelial cells recruit leukocytes by selectively expressing adhesion molecules on their surface following stimulation with inflammatory cytokines, such as TNFα and IL-1β, and certain reactive oxygen species (ROS). Adhesion molecules include vascular cell adhesion molecules (VCAM), intracellular adhesion molecules (ICAM) and endothelial cell selectin (E-selectin). Our laboratory, and others, has recently demonstrated that the AMP activated protein kinase (AMPK) regulates eNOS phosphorylation and NO synthesis in cultured endothelial cells, and this has been confirmed by the use of constitutively active and dominant negative mutant forms of AMPK. However, the molecular mechanisms and functional effects of AMPK activation in the endothelial remain poorly characterised. In this study we have utilised the AMPK activator AICAR and mutant AMPK adenoviral constructs to elucidate the effect of AMPK cascade activation on monocyte adhesion and adhesion molecule expression in human aortic endothelial cells (HAECs). Incubation with AICAR and infection with the constitutively active AMPK adenoviral construct was found to reduce U937 pro-monocyte adhesion to HAECs. This response appears to be bi-phasic, comprised of distinct short, NO-dependent (45min) and longer-term NO-independent (4hr) components. The NO-dependent reduction in monocyte adhesion was found to be independent of changes in cell surface expression of VCAM-1, ICAM-1 and E-Selectin adhesion molecules, while the longer term AICAR stimulation produced significant reductions in adhesion molecule expression. We propose that AMPK negatively regulates leukocyte adhesion in a bi-phasic manner, implying a role for AMPK in atherogenesis and vascular endothelial function.