This study presents evidence that phosphoinositide 3-kinase (PI3K) plays a concerted role with phospholipase Cγ in initiating antigen-mediated Ca2+ signaling in mast cells via a phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3)-sensitive Ca2+ entry pathway. Exogenous PI(3,4,5)P3 at concentrations close to its physiological level induces instantaneous Ca2+ influx into RBL-2H3 cells. This PI(3,4,5)P3-induced intracellular Ca2+ 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 Δp85 suppresses the Ca2+ response to the cross-linking of the high affinity receptor for IgE (FcεRI). Concomitant treatment of RBL-2H3 cells with LY294002 or Δp85 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)P3-sensitive Ca2+ entry and capacitative Ca2+ entry represent major Ca2+ influx pathways that sustain elevated [Ca2+]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 FcεRI-mediated Ca2+ response. Considering the underlying mechanism, our data suggest that PI(3,4,5)P3 directly stimulates a Ca2+ transport system in plasma membranes. Together, these data provide a molecular basis to account for the role of PI3K in the regulation of FcεRI-mediated degranulation in mast cells.