It has been known for a number of years that neutrophils and macrophages secrete H₂O₂while fighting disease, and the levels obtained within the vasculature under these conditions can reach several hundred micromolar. Because the effect of H₂O₂on vascular smooth muscle is not fully understood, the present study examined the cellular effects of H₂O₂on coronary arteries. Under normal ionic conditions, H₂O₂relaxed arteries that were precontracted with prostaglandin F_2α or histamine (EC₅₀ = 252 ± 22 μM). The effect of H₂O₂was concentration dependent and endothelium independent. In contrast, H₂O₂did not relax arteries contracted with 80 mM KCl, suggesting involvement of K⁺ channels. Single-channel patch-clamp recordings revealed that H₂O₂increased the activity of the large-conductance (119 pS), Ca²⁺- and voltage-activated K⁺(BK_Ca) channel. This response was mimicked by arachidonic acid and inhibited by eicosatriynoic acid, a lipoxygenase blocker, suggesting involvement of leukotrienes. Further studies on intact arteries demonstrated that eicosatriynoic acid not only blocked the vasodilatory response to H₂O₂but unmasked a vasoconstrictor effect that was reversed by blocking cyclooxygenase activity with indomethacin. These findings identify a novel effector molecule, the BK_Cachannel, which appears to mediate the vasodilatory effect of H₂O₂, and suggest that a single signaling pathway, arachidonic acid metabolism, can mediate the vasodilatory and vasoconstrictor effects of H₂O₂and possibly other reactive oxygen species.