Epoxyeicosatrienoic acids (EETs) are endothelium-derived cytochrome P-450 (CYP) metabolites of arachidonic acid that relax vascular smooth muscle by large- conductance calcium- activated potassium (BKCa) channel activation and membrane hyperpolarization. We hypothesized that if smooth muscle cells (SMCs) had the capacity to synthesize EETs, endogenous EET production would increase BKCa channel activity. Bovine coronary SMCs were transduced with adenovirus coding the CYP Bacillus megaterium -3 (F87V) (CYP BM-3) epoxygenase that metabolizes arachidonic acid exclusively to 14(S), 15(R)EET. Adenovirus containing the cytomegalovirus promoter- Escherichia coli beta-galactosidase was used as a control. With the use of an anti-CYP BM-3 (F87V) antibody, a 124-kDa 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 [C-14] 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(-5) M) increased 14,15-EET release twofold and BKCa channel activity fourfold in cell- attached patches. Methacholine-induced increases in BKCa channel activity were blocked by the CYP inhibitor 17-octadecynoic acid (10(-5) M). 14 S), 15(R)-EET was more potent than 14(R), 15(S)-EET in relaxing bovine coronary arteries and activating BKCa 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 BKCa channels. These studies indicate that 14(S), 15(R)-EET is a sufficient endogenous activator of BKCa channels in coronary SMCs.