AMP-activated protein kinase (AMPK) is the downstream component of a protein kinase cascade important in the regulation of cellular and whole body metabolism. Phosphorylation of Thr172 on the catalytic α subunit by an upstream AMPK kinase is essential for AMPK activity. The tumour suppressor LKB1 and Ca²⁺ /calmodulin dependent protein kinase kinase (CaMKK) have both been identified as upstream kinases that phosphorylate this residue. LKB1 responds to an increase in the cellular AMP:ATP ratio whereas CaMKK acts in a Ca²⁺ -dependent, AMP–independent manner. Evidence suggests endothelial dysfunction, largely characterised by reduced nitric oxide (NO) bioavailability, is an important feature in regards to the development of type 2 diabetes and cardiovascular disease. AMPK has been shown to mediate some physiological responses in the endothelium and its activation may have beneficial effects in the dysfunctional endothelium. Vascular endothelial growth factor (VEGF) is an important regulator of endothelial cell function. VEGF stimulates growth and promotes survival of endothelial cells, stimulates NO production and vasodilation and induces angiogenesis. VEGF has been shown to increase intracellular Ca²⁺ levels in endothelial cells, an effect dependent on PLCγ activation. Subsequent to PLC activation DAG and IP₃ are produced. DAG increases Ca²⁺ influx through non-selective cation channels, whereas IP₃ promotes the release of Ca²⁺ from intracellular stores. Previous studies in our laboratory have shown that VEGF is a novel activator of AMPK. The aim of the present study was to investigate the signalling mechanisms leading to AMPK activation and the functional relevance of AMPK in endothelial cells stimulated with VEGF. As VEGF can increase intracellular Ca²⁺ levels we wanted to investigate whether CaMKK plays a role in VEGF mediated AMPK activity. Human aortic endothelial cells (HAECs) treated with VEGF are phosphorylated at Thr172 resulting in a 2-fold increase in AMPK activity. These effects are dependent on CaMKK as demonstrated by the selective inhibitor STO-609. High potassium induced depolarisation used to reduce the driving force for Ca²⁺ entry through non-selective cation channels in HAECs abolished VEGF mediated AMPK activity. Inhibition of IP₃ sensitive intracellular stores with 2-APB had no effect. We propose that VEGF increases Ca²⁺ influx through non-selective cation channels resulting in stimulation of CaMKK and subsequently an increase in AMPK activity.