ABSTRACT Over-expressing calcineurin (CLN) in the mouse heart induces severe hypertrophy that progresses to heart failure, providing an opportunity to define the relationship between energetics and contractile performance in the severely failing mouse heart. Contractile performance was studied in isolated hearts at different pacing frequencies and during dobutamine challenge. Energetics were assessed by 31P NMR spectroscopy as [ATP], [phosphocreatine] (PCr) and the free energy of ATP hydrolysis (|G_~ATP|). Mitochondrial and glycolytic enzyme activities, myocardial oxygen consumption and myocyte ultrastructure were determined. In transgenic (TG) hearts under all work states, indices of systolic performance were reduced and [ATP] and capacity for ATP synthesis were lower compared to non-transgenic (NTG) hearts. This is the first report showing that myocardial [ATP] is lower in a transgenic mouse model of heart failure. [PCr] was also lower, despite an unexpected increase in the total creatine pool. Because [Pi] remained low despite lower [ATP] and [PCr], the free energy available from ATP hydrolysis was normal; however, chemical energy was not transduced into systolic performance. This was most apparent with -adrenergic stimulation of TG hearts, in which, for similar changes in |G_~ATP|, decreased rather than increased systolic pressure was observed. Structural abnormalities observed for both sarcomeres and mitochondria likely contribute to decreased contractile performance. Based on the increases in enzyme activities of proteins important for ATP supply observed after treatment with the CLN inhibitor, cyclosporin A (CsA), we also conclude that there is a CLN-directed inhibition of ATP producing pathways in both NTG and TG hearts.