Nitric oxide (NO)‐mediated activation of the cGMP‐dependent protein kinase (cGK) leads to relaxation of vascular smooth muscle (VSM) through a process involving phosphorylation of the BK channel. In VSM cells, this modification increases channel open probability, resulting in membrane hyperpolarization and closure of voltage‐gated Ca2+ channels. Although this paradigm is now generally accepted, the target residues of cGK‐mediated phosphorylation on the BK channel have thus far been inadequately characterized at the molecular level. We have scrutinized this BK channel phosphorylation by identifying several putative phosphorylation sites on the C‐terminus via electrophysiological and biochemical assays. Wild type (WT) and Ser to Ala mutated BK channels were expressed in cultured rat aortic VSM cells (A7r5) and studied using the cell‐attached patch clamp technique. The NO donor spermine NONOate (5 μM) and the membrane‐permeant analogue db‐cGMP (0.1 mM) enhanced WT BK channel activity, while several BK channel mutants had diminished responses to either stimulant. Exposure to the Ca2+ ionophore A23187 enhanced channel activity in both WT and mutant channels. These results demonstrate that BK current enhancement by NO/cGK‐mediated phosphorylation requires multiple sites on the C‐terminus tail, in an apparent all‐or‐none fashion. Supported by NSERC (APB) and a Kertland Family Scholarship (BDK).