This study presents evidence that phosphoinositide 3-kinase (PI3K) plays a concerted role with phospholipase Cgamma in initiating antigen-mediated Ca(2+) signaling in mast cells via a phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P(3))-sensitive Ca(2+) entry pathway. Exogenous PI(3,4,5)P(3) at concentrations close to its physiological level induces instantaneous Ca(2+) influx into RBL-2H3 cells. This PI(3,4,5)P(3)-induced intracellular Ca(2+) increase is independent of phospholipase C activity or the depletion of internal stores. Moreover, inhibition of PI3K by LY294002 or by overexpression of the dominant negative inhibitor Deltap85 suppresses the Ca(2+) response to the cross-linking of the high affinity receptor for IgE (FcepsilonRI). Concomitant treatment of RBL-2H3 cells with LY294002 or Deltap85 and 2-aminoethyl diphenylborate, a cell-permeant antagonist of D-myo-inositol 1,4,5-trisphosphate receptors, abrogates antigen-induced Ca(2+) signals, whereas either treatment alone gives rise to partial inhibition. Conceivably, PI(3,4,5)P(3)-sensitive Ca(2+) entry and capacitative Ca(2+) entry represent major Ca(2+) influx pathways that sustain elevated [Ca(2+)]i to achieve optimal physiological responses. This study also refutes the second messenger role of D-myo-inositol 1,3,4,5-tetrakisphosphate in regulating FcepsilonRI-mediated Ca(2+) response. Considering the underlying mechanism, our data suggest that PI(3,4,5)P(3) directly stimulates a Ca(2+) transport system in plasma membranes. Together, these data provide a molecular basis to account for the role of PI3K in the regulation of FcepsilonRI-mediated degranulation in mast cells.