A variety of mammalian cells undergo apoptosis when deprived of growth factors, but the intracellular signaling pathways responsible for cell survival remain to be characterized. In the present study, we have investigated the role of PI 3-kinase and pp70S6K in growth factor-dependent survival of rodent fibroblasts. As previously reported for PC12 pheochromocytoma cells, Rat-1 and REF52 cells underwent apoptosis following either serum-deprivation or treatment with the PI 3-kinase inhibitors wortmannin and LY294002. In contrast, NIH3T3 and BALB 3T3 cells were resistant to apoptosis induced by either serum-deprivation or PI 3-kinase inhibition. It thus appears that PI 3-kinase is specifically required to prevent apoptosis of fibroblast cell lines that are dependent upon growth factors for their survival. Consistent with this role of PI 3-kinase, serum and growth factors maintained steady state levels of PI 3-kinase activity that rapidly decreased following serum-deprivation. Serum and growth factors similarly maintained a steady state level of pp70S6K, which is thought to be activated downstream of PI 3-kinase. However, inhibition of pp70S6K activation by rapamycin failed to induce apoptosis in either Rat-1 or PC12 cells. Cell survival thus appears to require a PI 3-kinase signaling pathway that is independent of pp70S6K activation.