Carbon monoxide (CO) is generated endogenously by the enzyme heme oxygenase. Although CO is a known vasodilator, cellular signaling mechanisms are poorly understood and are a source of controversy. The goal of the present study was to investigate mechanisms of CO dilation in porcine cerebral arterioles. Data indicate that exogenous or endogenously produced CO is a potent activator of large-conductance Ca2+-activated K+ (K(Ca)) channels and Ca2+ spark-induced transient K(Ca) currents in arteriole smooth muscle cells. In contrast, CO is a relatively poor activator of Ca2+ sparks. To understand the apparent discrepancy between potent effects on transient K(Ca) currents and weak effects on Ca2+ sparks, regulation of the coupling relationship between these events by CO was investigated. CO increased the percentage of Ca2+ sparks that activated a transient K(Ca) current (ie, the coupling ratio) from approximately 62% in the control condition to 100% and elevated the slope of the amplitude correlation between these events approximately 2.6-fold, indicating that Ca2+ sparks induced larger amplitude transient K(Ca) currents in the presence of CO. This signaling pathway for CO is physiologically relevant because ryanodine, a ryanodine-sensitive Ca2+ release channel blocker that inhibits Ca2+ sparks, abolished CO dilation of pial arterioles in vivo. Thus, CO dilates cerebral arterioles by priming K(Ca) channels for activation by Ca2+ sparks. This study presents a novel dilatory signaling pathway for CO in the cerebral circulation and appears to be the first demonstration [corrected] of a vasodilator that acts by increasing the effective coupling of Ca2+ sparks to K(Ca) channels.