Doxorubicin (DOX) is a widely used chemotherapeutic drug for human hepatocellular carcinoma (HCC). A major limitation to its effectiveness is the development of multidrug resistance of cancer cells. In clinical trials, patients with advanced HCC were treated with high-dose acetaminophen (HAAP) in an effort to improve the antitumor activity of chemotherapeutics. In this study, we investigated the effect of concomitant treatment of DOX and HAAP on hepatoma-derived HepG2 cells. Viability, cell cycle distribution, and ultrastructure were examined. Unexpectedly, HAAP, when added to DOX-exposed cells, increased cell viability, released cell cycle arrest, and decreased apoptosis. To elucidate the mechanisms by which HAAP reduces the DOX lethal effect to HepG2 cells, we investigated the multidrug resistance P-glycoprotein (P-gp) and p44/42-mitogen-activated protein kinase (MAPK) pathways. The P-gp function was enhanced by DOX and HAAP, and it was further stimulated during combined treatment, leading to decreased DOX retention. Verapamil (VRP), when added to DOX ϩ HAAP exposure, increased DOX accumulation and restored DOXinduced toxicity. The increased phospho-p44/42-MAPK level in DOX-exposed cells was inhibited by HAAP. In addition, suppression of p44/42 activation by the p44/42-MAPK inhibitor 2Ј-amino-3Ј-methoxyflavone (PD98059) blocked DOX-induced apoptosis. These findings suggest that the antagonistic effect of concomitant DOX ϩ HAAP treatment occurs as a result of interactive stimulation of P-gp, generating decreased intracellular drug concentrations. Furthermore, inhibition of the p44/ 42-MAPK phosphorylation by HAAP could abolish the DOXinduced cell death pathway. Thus, combined treatment by DOX ϩ HAAP, intended to improve chemotherapeutic efficacy, could have an opposite effect facilitating cancer cell survival.