Metformin, a drug widely used to treat type 2 diabetes, was recently shown to activate the AMP-activated protein kinase (AMPK) in intact cells and in vivo. In this study we addressed the mechanism for this effect. In intact cells, metformin stimulated phosphorylation of the key regulatory site (Thr-172) on the catalytic (alpha) subunit of AMPK. It did not affect phosphorylation of this site by either of two upstream kinases in cell-free assays, although we were able to detect an increase in upstream kinase activity in extracts of metformin-treated cells. Metformin has been reported to be an inhibitor of complex 1 of the respiratory chain, but we present evidence that activation of AMPK in two different cell types is not a consequence of depletion of cellular energy charge via this mechanism. Whereas we have not established the definitive mechanism by which metformin activates AMPK, our results show that the mechanism is different from that of the existing AMPK-activating agent, 5-aminoimidazole-4-carboxamide (AICA) riboside. Metformin therefore represents a useful new tool to study the consequences of AMPK activation in intact cells and in vivo. Our results also show that AMPK can be activated by mechanisms other than changes in the cellular AMP-to-ATP ratio.