Signaling by phosphatidylinositol (PI) 3-kinases is mediated by 3- phosphoinositides, which bind to Pleckstrin homology (PH) domains that are present in a wide spectrum of proteins. PH domains can be classified into three groups based on their different lipid binding specificities. Distinct 3-phosphoinositides can accumulate upon PI 3-kinase activation in cells in response to different stimuli and mediate specific cellular responses. In Swiss 3T3 mouse fibroblasts, oxidative stress induced by 1 mM H2O2 caused almost exclusive accumulation of phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2), whereas osmotic stress increased both phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) and PtdIns(3,4)P2 levels. The increase in PtdIns(3,4)P2 levels, caused by oxidative stress, correlated with the activation of protein kinase B, which has a promiscuous PH domain that binds both PtdIns(3,4,5)P3 and PtdIns(3,4)P2. p70 S6 kinase, another signaling component downstream of PI 3-kinase, however, was not activated by this oxidative stress-induced increase in PtdIns(3,4)P2 levels. Increased PtdIns(3,4,5)P3 and PtdIns(3,4)P2 levels in response to osmotic stress did not correlate with protein kinase B activation, because of concomitant activation of an inhibitory pathway, but p70 S6 kinase was activated by osmotic stress. These results demonstrate that PtdIns(3,4)P2 can accumulate independently of PtdIns(3,4,5)P3 and exerts a pattern of cellular responses that is distinct from that induced by accumulation of PtdIns(3,4,5)P3.