AMPK is a sensor of cellular energy status and a promising target for drugs aimed at metabolic disorders. We have studied the selectivity and mechanism of a recently described activator, C2, and its cell-permeable prodrug, C13. C2 was a potent allosteric activator of a1-complexes that, like AMP, also protected against Thr172 dephosphorylation. Compared with AMP, C2 caused only partial allosteric activation of a2-complexes and failed to protect them against dephosphorylation. We show that both effects could be fully restored by exchanging part of the linker between the autoinhibitory and C-terminal domains in a2, containing the equivalent region from a1 thought to interact with AMP bound in site 3 of the ? subunit. Consistent with our results in cell-free assays, C13 potently inhibited lipid synthesis in hepatocytes from wild-type and was largely ineffective in AMPK-knockout hepatocytes; its effects were more severely affected by knockout of a1 than of a2, ß1, or ß2.