The g subunits of heterotrimeric AMPK complexes contain the binding sites for the regulatory adenine nucleotides AMP, ADP and ATP. We addressed whether complexes containing different g isoforms display different responses to adenine nucleotides by generating cells stably expressing FLAG-tagged versions of the g1, g2 or g3 isoform. When assayed at a physiological ATP concentration (5 mM), g1- and g2-containing complexes were allosterically activated almost 10-fold by AMP, with EC50 values one to two orders of magnitude lower than the ATP concentration. By contrast, g3 complexes were barely activated by AMP under these conditions, although we did observe some activation at lower ATP concentrations. Despite this, all three complexes were activated, due to increased Thr172 phosphorylation, when cells were incubated with mitochondrial inhibitors that increase cellular AMP. With g1 complexes, activation and Thr172 phosphorylation induced by the upstream kinase LKB1 (but not CaMKKb) in cell-free assays was markedly promoted by AMP and, to a smaller extent and less potently, by ADP. However, effects of AMP or ADP on activation and phosphorylation of the g2 and g3 complexes were small or insignificant. Binding of AMP or ADP protected all three g subunit complexes against inactivation by Thr172 dephosphorylation; with g2 complexes, ADP had similar potency to AMP, but with g1 and g3 complexes ADP was less potent than AMP. Thus, AMPK complexes containing different g subunit isoforms respond differently to changes in AMP, ADP or ATP. These differences may tune the responses of the isoforms to fit their differing physiological roles.