Large-conductance Ca2+-activated K+ channels (BK channels) are highly expressed in human glioma cells. However, less is known about their biological function in these cells. We used the patch-clamp technique to investigate activation properties of BK channels and time-lapse microscopy to evaluate the role of BK channel activation in migration of 1321N1 human glioma cells. In whole cells, internal perfusion with a solution containing 500nM free Ca2+ and external application of the BK channel opener phloretin (100M) shifted the activation threshold of BK channel currents toward more negative voltages of about 30mV, which is close to the resting potential of the cells. The concentration of intracellular Ca2+ in fura-2-loaded 1321N1 cells was measured to be 23519nM and was increased to 47225nM after treatment with phloretin. Phloretin and another BK channel opener NS1619 (100M) reduced the migration velocity by about 50%. A similar reduction was observed following muscarinic stimulation of glioma cells with acetylcholine (100M). The effects of phloretin, NS1619 and acetylcholine on cell migration were completely abolished by co-application of the specific BK channel blockers paxilline (5M) and iberiotoxin (100nM). The phloretin-induced increase in intracellular Ca2+ was unaffected by the removal of extracellular Ca2+ and co-application of paxilline. These findings indicate that glioma cell migration was inhibited through BK channel activation, independent of intracellular Ca2+.