The p53 tumor suppressor plays a key role in the natural protection against cancer. Activation of p53 by DNA-damaging agents can contribute to successful elimination of cancer cells via chemotherapy-induced apoptosis. The phosphatidylinositol-3 kinase (PI3K) pathway, triggered in normal cells upon exposure to growth factors, regulates a cascade of proliferation and survival signals. The PI3K pathway is abnormally active in many cancers, thus making it an attractive target for inactivation in an attempt to achieve better cancer therapy. We report here that exposure to LY294002, a potent PI3K inhibitor, aborts the activation of p53 by several drugs commonly used in cancer chemotherapy. Concomitantly, LY294002 attenuates p53-dependent, chemotherapy-induced apoptosis of cancer cells. These findings invoke an unexpected positive role for PI3K in p53 activation by anticancer agents, and suggest that the efficacy of PI3K inhibitors in cancer therapy may be greatly affected by the tumor p53 status.