Myocardial contractility depends on Ca2+ release from and uptake into the sarcoplasmic reticulum (SR). The Ca2+ gradient between the SR matrix and the cytosol (SR Ca2+ gradient) is maintained by the SR Ca2+-ATPase using the free energy available from hydrolysis of ATP. The activity of the SR Ca2+-ATPase is not only dependent on the energy state of the cell but is also kinetically regulated by SR proteins such as phospholamban. To evaluate the importance of thermodynamic and kinetic regulation of the SR Ca2+ gradient, we examined the relationship between the energy available from ATP hydrolysis (DeltaGATP) and the energy required for maintenance of the SR Ca2+ gradient (DeltaGCa2+SR) during physiological and pathological manipulations that alter DeltaGATP and the phosphorylation state of phospholamban. We used our previously developed 19F nuclear magnetic resonance method to measure the ionized [Ca2+] in the SR of Langendorff-perfused rabbit hearts. We found that addition of either pyruvate or isoproterenol resulted in an increase in left ventricular developed pressure and an increase in [Ca2+]SR. Pyruvate increased DeltaGATP, and the increase in the SR Ca2+ gradient was matched to the increase in DeltaGATP; DeltaGATP increased from 58.3+/-0.5 to 60.4+/-1.0 kJ/mol (P<0.05), and DeltaGCa2+SR increased from 47.1+/-0.3 to 48.5+/-0.1 kJ/mol (P<0.05). In contrast, the increase in the SR Ca2+ gradient in the presence of isoproterenol occurred despite a decline in DeltaGATP from 58. 3+/-0.5 to 55.8+/-0.6 kJ/mol. Thus, the data indicate that the SR Ca2+ gradient can be increased by an increase in DeltaGATP, and that the positive inotropic effect of pyruvate can be explained by improved energy-linked SR Ca2+ handling, whereas the results with isoproterenol are consistent with removal of the kinetic limitation of phospholamban on the activity of the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase, which allows the SR Ca2+ gradient to move closer to its thermodynamic limit. Ischemia decreases DeltaGATP, and this should also have an effect on SR Ca2+ handling. During 30 minutes of ischemia, DeltaGATP decreased by 12 kJ/mol, but the decrease in DeltaGCa2+SR was 16 kJ/mol, greater than would be predicted by the fall in DeltaGATP and consistent with increased SR Ca2+ release and increased SR Ca2+ cycling. Because ischemic preconditioning is reported to decrease SR Ca2+ cycling during a subsequent sustained period of ischemia, we examined whether ischemic preconditioning affects the relationship between the fall in DeltaGATP and the fall in DeltaGCa2+SR during ischemia. We found that preconditioning attenuated the fall in DeltaGCa2+SR during ischemia; the fall in DeltaGCa2+SR was of comparable magnitude to the fall in DeltaGATP, and this was associated with a significant improvement in functional recovery during reperfusion. The data suggest that there is both thermodynamic regulation of the SR Ca2+ gradient by DeltaGATP and kinetic regulation, which can alter the relationship between DeltaGATP and DeltaGCa2+SR.