In addition to the voltage-gated K⁺ current (I_Kv) and Ca²⁺-activated K⁺ current (I_KCa) that are commonly expressed in various lymphocytes, the immature B cell line (WEHI-231) and mouse splenic B cells express phosphatidylinositol 4,5-bisphosphate (PIP₂)-inhibited large conductance background K⁺ channels (LK_bg). The activity of LK_bg is low in cell-attached conditions. After making inside-out patches, LK_bg is spontaneously activated while inhibited by the application of ATP via PIP₂ formation. In the immature B lymphocytes, generation of arachidonic acid (AA) by phospholipase A₂ has been suggested to be involved in the apoptosis and growth arrest by antigen receptor stimulation. In this study, effects of AA on the ion channels were investigated in WEHI-231 cells, mainly focusing on LK_bg. The application of AA initially suppressed I_Kv and I_KCa while subsequently increased background-type K⁺ currents. In single channel recordings, LK_bg were activated by AA and other unsaturated fatty acids, which became weaker as the acyl chain is more saturated; AA > oleic acid > plamitic acid. AA showed partial activating effects on LK_bg directly inhibited by PIP₂. The activation of LK_bg by AA was fully reversible and unaffected by the treatment with inhibitors of metabolizing enzymes for AA. Consistent with the activation of LK_bg, the application of AA induced strong hyperpolarization of WEHI-231 and a subgroup of mouse splenic B lymphocytes. The hyperpolarization by AA was resistant with charybdotoxin, a selective blocker for I_KCa. AA also activated small amplitudes of nonselective cationic current in WEHI-231 cells. The AA-induced activation of background K⁺ current and hyperpolarization was rarely observed in Bal-17, a mature B cell line of mice. In summary, the LK_bg highly expressed in the immature B lymphocytes are potently activated by AA while other types of K⁺ channels commonly expressed in mature B lymphocytes are suppressed. The strong hyperpolarization and efflux of electrolytes (KCl) with water might play a role in the decrease of cell volume during apoptosis in immature B lymphocytes.