p70 S6 kinase (p70S6K) plays an important role in protein translation and cell cycle progression. Increased levels of p70S6K have been associated with drug resistance. In this study, we have investigated the involvement of p70S6K in DNA damage-induced apoptosis. The DNA-damaging agent cisplatin caused a concentration-dependent decrease in the level of full-length p70S6K in small cell lung cancer H69 and non-small cell lung cancer A549 cells with a concomitant increase in the level of an approximately 45 kDa fragment. The proteolytic cleavage of p70S6K was inhibited by a broad specificity caspase inhibitor but not by the proteosome or calpain inhibitor. Cell-permeable peptide inhibitor and siRNA against caspase-3 inhibited cisplatin-induced proteolytic cleavage of p70S6K. In vitro-translated p70S6K was cleaved by human recombinant caspase-3. Cisplatin failed to induce cleavage of p70S6K in MCF-7 cells that lack functional caspase-3, but ectopic expression of caspase-3 in MCF-7 cells resulted in the cleavage of p70S6K. p70S6K was primarily cleaved at a noncanonical recognition site, Thr-Pro-Val-Asp, after Asp-393. Site-directed mutagenesis of Asp-393 to Ala resulted in protection against cisplatin-mediated apoptosis, whereas introduction of the N-terminal cleaved fragment resulted in potentiation of cisplatin-induced apoptosis. These results suggest that p70S6K is a novel substrate for caspase-3 and that the proteolytic cleavage of p70S6K is important for cisplatin-induced apoptosis.