Fatty acids are the main fuel for the myocardium in vivo. They increase oxygen consumption, but the regulation of their β-oxidation is not well known. Since Ca 2+ and matrix volume have been implicated in the regulation of fatty acid oxidation in liver mitochondria, we set out to investigate the effects of Ca 2+ on cellular respiration and energetics in the isolated perfused rat heart when oxidizing a short-chain fatty acid. Infusion of hexanoate increased oxygen consumption, while stepwise changes in the perfusate Ca 2+ concentration in the range 0.5–2.5 mm caused the mechanical work output and oxygen consumption to change in parallel. Hexanoate addition increased the cellular energy state as determined by 31 P NMR and evaluated from the cytosolic [ATP]/[ADP]·[Pi] ratio. During fatty acid infusion the energy state decreased slightly upon Ca 2+ -induced inotropy, and after discontinuation of the hexanoate infusion the de-energization was more pronounced. The fatty acid caused an extensive partially reversible reduction of flavoproteins and NAD with a slight tendency for oxidation during Ca 2+ -induced inotropy. The data are in agreement with the notion that oxygen consumption during fatty acid oxidation is mainly determined by the energy expenditure, even in the presence of Ca 2+ -induced alterations in the inotropic state. The constancy of the redox states of mitochondrial flavins and NADH/NAD during large changes in oxygen consumption is interpreted as indicating stabilization of the mitochondrial redox states by Ca 2+ -linked regulation.