—Large-conductance potassium (BK) channels in vascular smooth muscle cells (VSMCs) sense both changes in membrane potential and in intracellular Ca²⁺ concentration. BK channels may serve as negative feedback regulators of vascular tone by linking membrane depolarization and local increases in intracellular Ca²⁺ concentration (Ca²⁺ sparks) to repolarizing spontaneous transient outward K⁺ currents (STOCs). BK channels are composed of channel-forming BKα and auxiliary BKβ1 subunits, which confer to BK channels an increased sensitivity for changes in membrane potential and Ca²⁺. To assess the in vivo functions of this β subunit, mice with a disrupted BKβ1 gene were generated. Cerebral artery VSMCs from BKβ1 −/− mice generated Ca²⁺ sparks of normal amplitude and frequency, but STOC frequencies were largely reduced at physiological membrane potentials. Our results indicate that BKβ1 −/− mice have an abnormal Ca²⁺ spark/STOC coupling that is shifted to more depolarized potentials. Thoracic aortic rings from BKβ1 −/− mice responded to agonist and elevated KCl with an increased contractility. BKβ1 −/− mice had higher systemic blood pressure than BKβ1 +/+ mice but responded normally to α₁-adrenergic vasoconstriction and nitric oxide–mediated vasodilation. We propose that the elevated blood pressure in BKβ1 −/− mice serves to normalize Ca²⁺ spark/STOC coupling for regulating myogenic tone. The full text of this article is available at http://www.circresaha.org.