Glioma cells prominently express a unique splice variant of a large conductance, calcium-activated potassium channel (BK channel). These channels transduce changes in intracellular calcium to changes of K(+) conductance in the cells and have been implicated in growth control of normal and malignant cells. The Ca(2+) increase that facilitates channel activation is thought to occur via activation of intracellular calcium release pathways or influx of calcium through Ca(2+)-permeable ion channels. We show here that BK channel activation involves the activation of inositol 1,4,5-triphosphate receptors (IP(3)R), which localize near BK channels in specialized membrane domains called lipid rafts. Disruption of lipid rafts with methyl-beta-cyclodextrin disrupts the functional association of BK channel and calcium source resulting in a >50% reduction in K(+) conductance mediated by BK channels. The reduction of BK current by lipid raft disruption was overcome by the global elevation of intracellular calcium through inclusion of 750 nm Ca(2+) in the pipette solution, indicating that neither the calcium sensitivity of the channel nor their overall number was altered. Additionally, pretreatment of glioma cells with 2-aminoethoxydiphenyl borate to inhibit IP(3)Rs negated the effect of methyl-beta-cyclodextrin, providing further support that IP(3)Rs are the calcium source for BK channels. Taken together, these data suggest a privileged association of BK channels in lipid raft domains and provide evidence for a novel coupling of these Ca(2+)-sensitive channels to their second messenger source.