T he molecular mechanisms underlying the effects of nitric oxide (NO) and carbon monoxide (CO), individually and collectively, on large-conductance calcium-activated K⁺ (K_Ca) channels were investigated in rat vascular smooth muscle cells (SMCs). Both NO and CO increased the activity of native K_Ca channels. Dehydrosoyasaponin-I, a specific agonist for β subunit of K_Ca channels, increased the open probability of native K_Ca channels only when it was delivered to the cytoplasmic surface of membrane. CO, but not NO, further increased the activity of native K_Ca channels that had been maximally stimulated by dehydrosoyasaponin-I. After treatment of SMCs with anti–K_Ca,β subunit antisense oligodeoxynucleotides, the stimulatory effect of NO, but not of CO, on K_Ca channels was nullified. CO, but not NO, enhanced the K_Ca current densities of heterologously expressed cloned K_Ca,α subunit, showing that the presence of K_Ca,β subunit is not a necessity for the effect of CO but essential for that of NO. Finally, pretreatment of SMCs with NO abolished the effects of subsequently applied CO or diethyl pyrocarbonate on K_Ca channels. In summary, the stimulatory effects of CO and NO on K_Ca channels rely on the specific interactions of these gases with K_Ca,α and K_Ca,β subunits.