Cellular nitric oxide (NO) and calcium levels have been reported to increase during various pathologies, including particularly ischaemia. In this study, we investigated whether elevated NO and calcium levels can synergistically damage isolated rat heart mitochondria. We found that NO and calcium together inhibited the oxygen consumption of mitochondria respiring on pyruvate + malate, but not mitochondria respiring on succinate. In the same conditions, complex I activity was synergistically inhibited by NO and calcium, and this inhibition was completely prevented by superoxide dismutase or urate, suggesting that the inhibition was mediated by peroxynitrite. Indeed, we found NO and calcium-stimulated mitochondrial production of peroxynitrite. The inhibition of complex I activity by NO and calcium was reversed by reduced thiols or light (as was complex I inhibition by S-nitrosothiols or peroxynitrite) suggesting that the inhibition may involve S-nitrosation or Fe-nitrosylation of complex I. However, NO and calcium also caused loss of mitochondrial cytochrome c, and the induced inhibition of respiration was partially reversed by addition of exogenous cytochrome c. Thus, NO and calcium appear to synergistically inhibit mitochondrial respiration, partly by inactivation of complex I and partly by inducing cytochrome c release.