Large conductance, calcium- and voltage-gated potassium (BK) channels are ubiquitous and critical for neuronal function, immunity, and smooth muscle contractility. BK channels are thought to be regulated by phosphatidylinositol 4,5-bisphosphate (PIP2) only through phospholipase C (PLC)–generated PIP2 metabolites that target Ca2+ stores and protein kinase C and, eventually, the BK channel. Here, we report that PIP2 activates BK channels independently of PIP2 metabolites. PIP2 enhances Ca2+-driven gating and alters both open and closed channel distributions without affecting voltage gating and unitary conductance. Recovery from activation was strongly dependent on PIP2 acyl chain length, with channels exposed to water-soluble diC4 and diC8 showing much faster recovery than those exposed to PIP2 (diC16). The PIP2–channel interaction requires negative charge and the inositol moiety in the phospholipid headgroup, and the sequence RKK in the S6–S7 cytosolic linker of the BK channel-forming (cbv1) subunit. PIP2-induced activation is drastically potentiated by accessory β1 (but not β4) channel subunits. Moreover, PIP2 robustly activates BK channels in vascular myocytes, where β1 subunits are abundantly expressed, but not in skeletal myocytes, where these subunits are barely detectable. These data demonstrate that the final PIP2 effect is determined by channel accessory subunits, and such mechanism is subunit specific. In HEK293 cells, cotransfection of cbv1+β1 and PI4-kinaseIIα robustly activates BK channels, suggesting a role for endogenous PIP2 in modulating channel activity. Indeed, in membrane patches excised from vascular myocytes, BK channel activity runs down and Mg-ATP recovers it, this recovery being abolished by PIP2 antibodies applied to the cytosolic membrane surface. Moreover, in intact arterial myocytes under physiological conditions, PLC inhibition on top of blockade of downstream signaling leads to drastic BK channel activation. Finally, pharmacological treatment that raises PIP2 levels and activates BK channels dilates de-endothelized arteries that regulate cerebral blood flow. These data indicate that endogenous PIP2 directly activates vascular myocyte BK channels to control vascular tone.