AMP-activated kinase (AMPK) controls cell energy homeostasis by modulating ATP synthesis and expenditure. In vitro studies have suggested AMPK may also control key elements of renal epithelial electrolyte transport but in vivo physiological confirmation is still insufficient. We studied sodium renal handling and extracellular volume regulation in mice with genetic deletion of AMPK catalytic subunits. AMPK alpha-1 KO mice exhibit normal renal sodium handling and a moderate antidiuretic state. This is accompanied by higher urinary aldosterone excretion rates and reduced blood pressure. Plasma volume, however, was found to be increased compared to wild-type mice. Thus blood volume is preserved despite a significantly lower hematocrit. The lack of a defect in renal function in AMPK alpha-1 KO mice could be explained by a compensatory upregulation in AMPK alpha-2 subunit. Therefore we used the Cre-loxP system to knock down AMPK alpha-2 expression in renal epithelial cells. Combining this approach with the systemic deletion of AMPK alpha-1 we achieved reduced renal AMPK activity, accompanied by a shift to a moderate water and salt wasting phenotype. Thus we confirm the physiologically relevant role of AMPK in the kidney. Furthermore, our results indicate that in vivo AMPK activity stimulates renal sodium and water reabsorption.