Crosslinking of IgE receptors by antigen initiates Ca2+ mobilization in mast cells by activating phospholipase-Cγ-mediated hydrolysis of phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2]. The resulting inositol 1,4,5-trisphosphate-mediated Ca2+ release from the endoplasmic reticulum (ER) activates store-operated Ca2+ entry, which is necessary for exocytotic release of inflammatory mediators. To investigate roles for PtdIns(4,5)P2- synthesizing isozymes of the type I phosphatidylinositol 4-phosphate 5-kinase family (PIP5K-I) in mast cell signaling, we compared the ectopic expression of wild-type and catalytically inactive PIP5K- Iβ in RBL-2H3 mast cells. Surprisingly, both antigen and thapsigargin-stimulated Ca2+ influx were reduced by overexpression of active PIP5K-Iβ, whereas antigen-stimulated Ca2+ release from ER stores was unaffected. Consistent with these results, Ca2+ entry stimulated by antigen or thapsigargin was enhanced by expression of a plasma-membrane-associated inositol polyphosphate 5′-phosphatase, whereas antigen-stimulated Ca2+ release from stores was reduced. To investigate the role of PIP5K-Iγ in antigen-stimulated Ca2+ mobilization, we used bone-marrow-derived mast cells from PIP5K- Iγ-/- mice. Antigen-stimulated Ca2+ release from ER stores was substantially reduced in the absence of PIP5K-Iγ, but thapsigargin-mediated Ca2+ entry was unaffected. In summary, PIP5K-Iγ positively regulates antigen-stimulated Ca2+ release from ER stores, whereas PIP5K-Iβ negatively regulates store-operated Ca2+ entry, suggesting that these different PIP5K-I isoforms synthesize functionally distinct pools of PtdIns(4,5)P2 at the plasma membrane.