Both Ca2+ and free radicals (FR) are accumulated in temporarily ischemic myocardium and might cause reperfusion injury. Respiratory function measured by polarography of isolated heart mitochondria before or after the in vitro treatment with Ca2+ (1.2 microM) and/or FR showed that Ca2+ or FR per se showed no or weak effect on state 3, but cotreatment of Ca2+ and FR prominently deteriorated state 3, state 4 and RCI. The injury was speculated to occur at cytochrome c itself or its reductase from enzyme assay in the respiratory chain. Furthermore, contrary to the published data, the synergistic action was not mitigated by phospholipase A2 inhibitors (dibucaine, mepacrine), membrane stabilizers (lidocaine, coenzyme Q10), Ca entry blocker (verapamil) or superoxide dismutase, suggesting refractory to therapy.