The cardiotoxic effects of doxorubicin, a potent chemotherapeutic agent, have been linked to DNA damage, oxidative mitochondrial damage, and nuclear translocation of p53, but the exact molecular mechanisms causing p53 transactivation and doxorubicin-induced cardiomyopathy are not clear. The present study was carried out to determine if extracellular signal-regulated kinases (ERKs), which are known to be activated by DNA damaging agents, are responsible for doxorubicin-induced p53 activation and oxidative mitochondrial damage in H9c2 cells. Cell death was measured by TUNEL, Annexin-V FITC, activation of caspase-9 and -3, and cleavage of PARP. We found that doxorubicin produced cell death in H9c2 cells in a time-dependent manner, beginning at six hours, and these changes are associated decreased expression of Bcl-2, increases in Bax and PUMA expression, and collapse of mitochondria membrane potential. The changes in cell death and Bcl-2 family proteins, however, were preceded by earlier activation and nuclear translocation of ERKs, followed by increased phosphorylation at serine 15 and nuclear translocation of the phosphorylated p53. The functional importance of ERK1/2 and p53 in doxorubicin-induced toxicity was further demonstrated by the specific ERK inhibitor U0126 and p53 inhibitor PFT that abrogated the changes in Bcl-2 family proteins, and cell death produced by doxorubicin. U0126 blocked phosphorylation and nuclear translocation of both ERK1/2 and p53, while PFT blocked only the changes in p53. Doxorubicin and ERK inhibitors produced similar changes in ERK1/2/p53, PARP and caspase-3 in neonatal rat cultured cardiomyocytes. Thus, we conclude that ERK1/2 are functionally linked to p53 and that the ERK1/2-p53 cascade is the upstream signaling pathway responsible for doxorubicin-induced cardiac cell apoptosis. ERKs and p53 may be considered as novel therapeutic targets for the treatment of doxorubicin-induced cardiotoxicity.