Background: Calcium overload and reactive oxygen species can injure mitochondria during ischemia and reperfusion. We hypothesized that mitochondrial injury occurs during cardiac resuscitation causing cytochrome c release to the cytosol and bloodstream while activating apoptotic pathways. Methods and Results: Plasma cytochrome c was measured using reverse-phase high performance liquid chromatography and western immunoblotting in rats undergoing 4 or 8 minutes of untreated ventricular fibrillation and 8 minutes of closed-chest resuscitation followed by 240 minutes post-resuscitation hemodynamic observation. A sham group served as control. Plasma cytochrome c rose progressively attaining levels 10-fold higher than in sham rats at 240 minutes post-resuscitation (p < 0.01) despite reversal of whole-body ischemia (decreases in arterial lactate). Cytochrome c levels inversely correlated with left ventricular stroke work (r = -0.40, p = 0.02). Western immunoblotting of left ventricular tissue demonstrated increased levels of 17-kDa cleaved caspase-3 fragments in the cytosol. Plasma cytochrome c was then serially measured in 12 resuscitated rats until the rat had died or cytochrome c had returned to baseline. In 3 survivors, cytochrome c rose slightly without exceeding 2 µg/ml and returned to baseline within 96 hours. In 9 non-survivors, cytochrome c rose progressively attaining significantly higher maximal levels (4.6 ± 2.0 vs 1.6 ± 0.3 µg/ml; p = 0.029) and at faster rates (0.7 ± 0.5 vs 0.1 ± 0.1 µg/ml/hr; p = 0.046) than in survivors. Conclusion: Plasma cytochrome c may represent a novel in vivo marker of mitochondrial injury after resuscitation from cardiac arrest that relates inversely with survival outcome.