We have reported that epoxyeicosatrienoic acids (EETs), the cytochrome P450 (CYP) epoxygenase metabolites of arachidonic acid (AA), are potent sarcolemmal ATP-sensitive K+ (KATP) channel activators. However, activation of cardiac and vascular KATP channels by endogenously produced EETs under physiological intracellular conditions has not been demonstrated and direct comparison of the mechanisms whereby EETs activate the KATP channels in cardiac myocytes versus vascular smooth muscle cells has not been made. In this study, we examined the effects of AA on KATP channels in freshly isolated cardiac myocytes from rats, wild-type (WT) and transgenic mice overexpressing CYP2J2 cDNA, and mesenteric arterial smooth muscle cells from rats. We also compared the activation of cardiac and vascular KATP channels by extracellularly and intracellularly applied 11,12-EET. We found that 1 microm AA enhanced KATP channel activities in both cardiac and vascular smooth muscle cells, and the AA effects were inhibited by preincubation with CYP epoxygenase inhibitors. Baseline cardiac KATP current densities in CYP2J2 transgenic mice were 190% higher than those of WT mice, and both were reduced to similar levels by CYP epoxygenase inhibition. Western blot analysis showed that expression of Kir6.2 and SUR2A was similar between WT and CYP2J2 transgenic hearts. 11,12-EET (5 microm) applied intracellularly enhanced the KATP currents by 850% in cardiac myocytes, but had no effect in vascular smooth muscle cells. In contrast, 11,12-EET (5 microm) applied extracellularly increased KATP currents by 520% in mesenteric arterial smooth muscle cells, but by only 209% in cardiac myocytes. Preincubation with 100 microm m-iodobenzylguanidine or 5 microm myristoylated PKI amide did not alter the activation of cardiac KATP channels by 5 microm 11,12-EET, but significantly inhibited activation of vascular KATP channels. Moreover, EET only enhanced the inward component of cardiac KATP currents, but activated both the inward and outward components of vascular KATP currents. Our results indicate that endogenously derived CYP metabolites of AA potently activate cardiac and vascular KATP channels. EETs regulate cardiac electrophysiology and vascular tone by KATP channel activation, albeit through different mechanisms: the cardiac KATP channels are directly activated by EETs, whereas activation of the vascular KATP channels by EETs is protein kinase A dependent.