Little is known about the role of mitochondrial NADP+-isocitrate dehydrogenase (NADP+-ICDH) in the heart, where this enzyme shows its highest expression and activity. We tested the hypothesis that in the heart, NADP+-ICDH operates in the reverse direction of the citric acid cycle (CAC) and thereby may contribute to the fine regulation of CAC activity (Sazanov and Jackson, FEBS Lett344: 109–116, 1994). We documented a reverse flux through this enzyme in rat hearts perfused with the medium-chain fatty acid octanoate using [U-13C5]glutamate and mass isotopomer analysis of tissue citrate (Comte et al., J Biol Chem 272: 26117–26124, 1997). In this study, we assessed the significance of our previous finding by perfusing hearts with long-chain fatty acids and tested the effects of changes in O2supply. We showed that under all of these conditions citrate was enriched in an isotopomer containing five13C atoms. This isotopomer can only be explained by substrate flux through reversal of the NADP+-ICDH reaction, which is evaluated at 3–7% of flux through citrate synthase. Small variations in reversal fluxes induced by low-flow ischemia that mimicked hibernation occurred despite major changes in contractile function and O2consumption of the heart as well as citrate and succinate release rates and tissue levels. Our data show a reverse flux through NADP+-ICDH and support its hypothesized role in the fine regulation of CAC activity in the normoxic and O2-deprived heart.