Epoxyeicosatrienoic acids (EETs) are endothelium-derived arachidonic acid metabolites of cytochrome P450. They dilate coronary arteries, open K⁺ channels, and hyperpolarize vascular smooth muscles. However, the mechanisms of these smooth muscle actions remain unknown. This study examined the effects of EETs on the large-conductance Ca²⁺-activated K⁺ channel (K_Ca) in smooth muscle cells of small bovine coronary arteries. In cell-attached patch-clamp experiments, 11,12-EET produced a 0.5- to 10-fold increase in the activity of the K_Ca channels when added in concentrations of 1, 10, and 100 nmol/L. In the inside-out excised membrane patch mode, 11,12-EET was without effect on the activity of the K_Ca channel unless GTP (0.5 mmol/L) or GTP and ATP (1 mmol/L) were added to the bath solution. In the presence of GTP and ATP, the increase in the K_Ca channel activity with 11,12-EET in inside-out patches was comparable to that in cell-attached patches. This effect of 11,12-EET in inside-out patches was blocked by the addition of GDP-β-S (100 μmol/L). In outside-out patches, 11,12-EET also increased the K_Ca channel activity when GTP and ATP were added to the pipette solution. The addition of a specific anti-G_Sα antibody (100 nmol/L) in the pipette solution completely blocked the activation of the K_Ca channels induced by 11,12-EET. An anti-Gβγ or anti-G_iα antibody was without effect. We conclude that 11,12-EET activates the K_Ca channels by a G_Sα-mediated mechanism. This mechanism contributes to the effects of EETs as endothelium-derived hyperpolarizing factors to hyperpolarize and relax arterial smooth muscle.