In various types of cells mechanical stimuli on plasma membrane activate phospholipase C (PLCs). However, the regulation of ion channels via mechanosensitive degradation of phosphatidylinositol 4,5-bisphosphate (PIP₂) is not known yet. The mouse B cells express Large conductance background K⁺ channels (LK_bg) that are inhibited by PIP₂. In inside-out patch clamp studies, the application of MgATP (1mM) also inhibited LK_bg due to the generation of PIP₂ by PI-kinases. In the presence of MgATP, membrane stretch induced by negative pipette pressure activated LK_bg, which was antagonized by PIP₂ (>1 µM) or higher concentration of MgATP (5 mM). The inhibition by PIP₂ was partially reversible. However, the application of methyl- -cyclodextrin, a cholesterol scavenger disrupting lipid rafts, induced the full recovery of LK_bg activity and facilitated the activation by stretch. In cell-attached patches, LK_bg were activated by hypotonic swelling of B cells as well as by negative pressure. The mechano-activation of LK_bg was blocked by U73122, a PLC inhibitor. Neither actin depolymerization nor the inhibition of lipid phosphatase blocked the mechanical effects. Direct stimulation of PLC by m-3M3FBS or by cross-linking IgM- type B cell receptors activated LK_bg. Western blot analysis and confocal microscopy showed that the hypotonic swelling of WEHI-231 induces tyrosine phosphorylation of PLC2 and PIP₂ hydrolysis of plasma membrane. The time-dependence of PIP₂ hydrolysis and LK_bg activation were similar. The presence of LK_bg and their stretch-sensitivity were also proven in fresh isolated mice splenic B cells. From above results, we propose a novel mechanism of stretch-dependent ion channel activation, namely, that the degradation of PIP₂ caused by stretch-activated PLC releases LK_bg from the tonic inhibition by PIP₂.