In order to elucidate the role of the mitogen-activated protein kinases, including JNK, p38 MAPK and ERK, as well as the survival-associated PI3K/Akt signaling pathway, in the response to chemotherapy, we have conducted a comparative study regarding the effects of doxorubicin on these pathways. Doxorubicin was determined to elicit the apoptosis of NIH3T3 cells in a dose-dependent manner. Prior to cell death, both Akt and p38 MAPK were transiently activated, and subsequently inactivated almost wholly, whereas ERK and JNK evidenced sustained activations in response to the drug treatment. The inhibition of PI3K/Akt and p38 MAPK both accelerated and enhanced doxorubicin-induced apoptosis and ERK inhibition apparently exerted negative effect on apoptosis. The modulation of PI3K/Akt activation by treatment of LY294002 or expression of Akt mutants such as Akt-DN or Myr-Akt exerted a significant effect on the activation of ERK1/2. We also observed that PI3K/Akt and sustained ERK activation were associated intimately with the etoposide-induced apoptosis. Taken together, our results clearly suggest that the differential regulation of the PI3K/Akt, ERK1/2, and p38 MAPK signaling pathways are crucial in the context of DNA-damaging drug-induced apoptosis, and this has compelled us to propose that the sustained activation of ERK1/2 pathway may be generally involved in the apoptosis induced by anticancer DNA-damaging drugs, including doxorubicin and etoposide.