p53/56lyn and other src family tyrosine kinases become activated in monocytes treated with LPS. In a variety of systems, phosphatidylinositol 3-kinase (PI 3-kinase) is believed to be a downstream effector of tyrosine kinases, and activation of PI 3-kinase results in increased levels of D3-phosphorylated metabolites of phosphatidylinositol (Ptdlns). To examine whether LPS activates PI 3-kinase, freshly isolated human, peripheral blood monocytes were labeled in vitro with [32P]orthophosphate, and inositol phospholipids were detected after extraction and separation of lipids by TLC. Levels of PtdIns 3,4,5-trisphosphate (PtdIns 3,4,5-P3) were elevated within minutes of exposure of cells to LPS. Analysis of 32P-labeled lipid extracts of U937 cells by HPLC confirmed that levels of PtdIns 3,4,5-P3 increased rapidly following LPS treatment. Increased levels of PtdIns 3,4,5-P3 in LPS-treated cells resulted from an increase in the specific activity of PI 3-kinase. Thus, anti-PI 3-kinase immunoprecipitates prepared from unlabeled monocytes and assayed in an in vitro phosphorylation assay, using PtdIns as substrate, showed higher enzymatic activity when these were prepared from lysates of LPS-treated cells as compared with control cells. PI 3-kinase activity in immunoprecipitates was elevated as early as 2 min after LPS exposure and was dose dependent, with increased activity being observed at LPS concentrations as low as 10 pg/ml. Activation of PI 3-kinase involved signaling through the monocyte cell surface molecule CD14, since pretreatment of cells with Abs to CD14 abrogated LPS-induced increases in Ptdlns 3,4,5-P3. Immunoprecipitates of p53/56lyn from LPS-treated cells showed a time-dependent and transient increase in PI 3-kinase activity assayed in vitro, coordinate with activation of p53/56lyn indicating that LPS induces the association and simultaneous activation of these two enzymes in vivo. These findings indicate that monocytes respond to LPS with the rapid activation of PI 3-kinase, resulting in transient increases in levels of Ptdlns 3,4,5-P3. This process is CD14 dependent and involves the physical association of PI 3-kinase with activated p53/56lyn.