Doxorubicin (DOX) is a potent antitumor agent available; however its clinical use is limited because of its cardiotoxicity. Cell death is a key component in DOX-induced cardiotoxicity, but its mechanisms are elusive. Here, we explore the role of superoxide, nitric oxide (NO) and peroxynitrite in DOX-induced cell death using both in vivo and in vitro models of cardiotoxicity. Western blot, real-time PCR, immunohistochemistry, flow cytometry, fluorescent microscopy, and biochemical assays were used to determine markers of apoptosis/necrosis, sources of NO and superoxide and their production. Left ventricular function was measured by pressure-volume system. We demonstrate increase in myocardial apoptosis (caspase-3 cleavage/activity, cytochrom c release, TUNEL), iNOS expression, mitochondrial superoxide generation, 3-nitrotyrosine (NT) formation, MMP2/9 gene expression, PARP activation (without major changes in NOX1, NOX2, p22phox, p40phox, p47phox, p67phox, xanthine oxidase, eNOS, nNOS expressions), decrease in myocardial contractility, catalase and glutathione peroxidase activities five days following DOX treatment to mice. All these effects of DOX were markedly attenuated by peroxynitrite scavengers. Doxorubicin dose-dependently increased mitochondrial superoxide and NT generation, apoptosis/necrosis in cardiac-derived H9c2 cells. The DOX- or peroxynitrite-induced apoptosis/necrosis positively correlated with intracellular NT formation, and could be abolished by peroxynitrite scavengers. The DOX-induced cell death and NT formation were also attenuated by selective iNOS inhibitors or in iNOS knockout mice. Various NO donors when co-administered with DOX, but not alone, dramatically enhanced DOX-induced cell death with concomitant increased NT formation. The DOX-induced cell death was also attenuated by cell permeable SOD, but not by cell permeable catalase, the xanthine oxidase inhibitor allopurinol or the NADPH oxidase inhibitors apocynine or DPI. Thus, peroxynitrite is a major trigger of DOX-induced cell death both in vivo and in vivo, and the modulation of the pathways leading to its generation or its effective neutralization can be of significant therapeutic benefit.