Glycolysis increases in hypertrophied hearts but the mechanisms are unknown. We studied the regulation of glycolysis in hearts with pressure-overload LV hypertrophy (LVH), a model that showed marked increases in the rates of glycolysis (by 2-fold) and insulin-independent glucose uptake (by 3-fold). Although the V(max) of the key glycolytic enzymes was unchanged in this model, concentrations of free ADP, free AMP, inorganic phosphate (P(i)), and fructose-2,6-bisphosphate (F-2,6-P2), all activators of the rate-limiting enzyme phosphofructokinase (PFK), were increased (up to 10-fold). Concentrations of the inhibitors of PFK, ATP, citrate, and H+ were unaltered in LVH. Thus, our findings show that increased glucose entry and activation of the rate-limiting enzyme PFK both contribute to increased flux through the glycolytic pathway in hypertrophied hearts. Moreover, our results also suggest that these changes can be explained by increased intracellular free [ADP] and [AMP], due to decreased energy reserve in LVH, activating the AMP-activated protein kinase cascade. This, in turn, results in enhanced synthesis of F-2,6-P2 and increased sarcolemma localization of glucose transporters, leading to coordinated increases in glucose transport and activation of PFK.