K＋-Cl−-cotransport (KCC) has been reported to have various cellular functions, including proliferation and apoptosis of human cancer cells. However, the signal transduction pathways that control the activity of KCC are currently not well understood. In this study we investigated the possible role of phospholipase A2 (PLA2)-arachidonic acid (AA) signal in the regulatory mechanism of KCC activity. Exogenous application of AA significantly induced K＋ efflux in a dose-dependent manner, which was completely blocked by R-(＋)-[2-n-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H- inden-5-yl]oxy]acetic acid (DIOA), a specific KCC inhibitor. N-Ethylmaleimide (NEM), a KCC activator- induced K＋ efflux was significantly suppressed by bromoenol lactone (BEL), an inhibitor of the calcium- independent PLA2 (iPLA2), whereas it was not significantly altered by arachidonyl trifluoromethylketone (AACOCF3) and p-bromophenacyl bromide (BPB), inhibitors of the calcium-dependent cytosolic PLA2 (cPLA2) and the secretory PLA2 (sPLA2), respectively. NEM increased AA liberation in a dose- and time-dependent manner, which was markedly prevented only by BEL. In addition, the NEM- induced ROS generation was significantly reduced by DPI and BEL, whereas AACOCF3 and BPB did not have an influence. The NEM-induced KCC activation and ROS production was not significantly affected by treatment with indomethacin (Indo) and nordihydroguaiaretic acid (NDGA), selective inhibitors of cyclooxygenase (COX) and lipoxygenase (LOX), respectively. Treatment with 5,8,11,14- eicosatetraynoic acid (ETYA), a non-metabolizable analogue of AA, markedly produced ROS and activated the KCC. Collectively, these results suggest that iPLA2-AA signal may be essentially involved in the mechanism of ROS-mediated KCC activation in HepG2 cells.