Epoxyeicosatrienoic acids (EETs) are endothelium-derived cytochrome P450 (CYP) metabolites of arachidonic acid that relax vascular smooth muscle by calcium-activated potassium (BK_Ca) channel activation and membrane hyperpolarization. We hypothesized that if smooth muscle cells (SMCs) had the capacity to synthesize EETs, endogenous EET production would increase BK_Ca channel activity. Bovine coronary SMCs were transduced with adenovirus coding the CYP Bacillus megaterium (BM)-3 (F87V) epoxygenase that metabolizes arachidonic acid exclusively to 14(S),15(R)-EET. Adenovirus containing the cytomegalovirus promoter-Escherichia coli -galactosidase was used as a control. Using an anti-CYP BM-3 (F87V) antibody, a 124 kD immunoreactive protein was detected only in CYP BM-3 transduced cells. Protein expression increased with increasing amounts of virus. When CYP BM-3 transduced cells were incubated with ¹⁴C-arachidonic acid, HPLC analysis detected 14,15-dihydroxyeicosatrienoic acid (14,15-DHET) and 14,15-EET. The identity of 14,15-EET and 14,15-DHET was confirmed by mass spectrometry. In CYP BM-3 transduced cells, methacholine (10^-5M) increased 14,15-EET release 2-fold and BK_Ca channel activity 4-fold in cell-attached patches. Methacholine-induced increases in BK_Ca channel activity were blocked by the CYP inhibitor, octadecynoic acid (10^-5M). 14(S),15(R)-EET was more potent than 14(R),15(S)-EET in relaxing bovine coronary arteries and activating BK_Ca channels. Thus, CYP BM-3 adenoviral transduction confers SMCs with epoxygenase activity. These cells acquire the capacity to respond to the vasodilator agonist by synthesizing 14(S),15(R)-EET from endogenous arachidonic acid to activate BK_Ca channels. These studies indicate that 14(S),15(R)-EET is a sufficient endogenous activator of BK_Ca channels in coronary SMCs.