Here we tested the role of calcium influx factor (CIF) and calcium-independent phospholipase A(2) (iPLA(2)) in activation of Ca(2+) release-activated Ca(2+) (CRAC) channels and store-operated Ca(2+) entry in rat basophilic leukemia (RBL-2H3) cells. We demonstrate that 1) endogenous CIF production may be triggered by Ca(2+) release (net loss) as well as by simple buffering of free Ca(2+) within the stores, 2) a specific 82-kDa variant of iPLA(2)beta and its corresponding activity are present in membrane fraction of RBL cells, 3) exogenous CIF (extracted from other species) mimics the effects of endogenous CIF and activates iPLA(2)beta when applied to cell homogenates but not intact cells, 4) activation of I(CRAC) can be triggered in resting RBL cells by dialysis with exogenous CIF, 5) molecular or functional inhibition of iPLA(2)beta prevents activation of I(CRAC), which could be rescued by cell dialysis with a human recombinant iPLA(2)beta, 6) dependence of I(CRAC) on intracellular pH strictly follows pH dependence of iPLA(2)beta activity, and 7) (S)-BEL, a chiral enantiomer of suicidal substrate specific for iPLA(2)beta, could be effectively used for pharmacological inhibition of I(CRAC) and store-operated Ca(2+) entry. These findings validate and significantly advance our understanding of the CIF-iPLA(2)-dependent mechanism of activation of I(CRAC) and store-operated Ca(2+) entry.