To test the hypothesis that intracellular Ca2+ activation of large-conductance Ca2+-activated K+ (BK) channels involves the cytosolic form of phospholipase A2 (cPLA2), we first inhibited the expression of cPLA2 by treating GH3 cells with antisense oligonucleotides directed at the two possible translation start sites on cPLA2. Western blot analysis and a biochemical assay of cPLA2 activity showed marked inhibition of the expression of cPLA2 in antisense-treated cells. We then examined the effects of intracellular Ca2+ concentration ([Ca2+]i) on single BK channels from these cells. Open channel probability (Po) for the cells exposed to cPLA2 antisense oligonucleotides in 0.1 microM intracellular Ca2+ was significantly lower than in untreated or sense oligonucleotide-treated cells, but the voltage sensitivity did not change (measured as the slope of the Po-voltage relationship). In fact, a 1,000-fold increase in [Ca2+]i from 0.1 to 100 microM did not significantly increase Po in these cells, whereas BK channels from cells in the other treatment groups showed a normal Po-[Ca2+]i response. Finally, we examined the effect of exogenous arachidonic acid on the Po of BK channels from antisense-treated cells. Although arachidonic acid did significantly increase Po, it did so without restoring the [Ca2+]i sensitivity observed in untreated cells. We conclude that although [Ca2+]i does impart some basal activity to BK channels in GH3 cells, the steep Po-[Ca2+]i relationship that is characteristic of these channels involves cPLA2.