14,15-Epoxyeicosatrienoic acid (EET), a cytochrome P-450 epoxygenase product of arachidonic acid (AA), reduced PGE₂formation by 40–75% in porcine aortic and murine brain microvascular smooth muscle cells. The inhibition was reversed 6–10 h after removal of 14,15-EET from the medium and was regioisomeric specific; 8,9-EET produced a smaller effect, whereas 11,12- and 5,6-EET were ineffective. Although the cells converted 14,15-EET to 14,15-dihydroxyeicosatrienoic acid (14,15-DHET), 14,15-DHET did not inhibit PGE₂formation, and the 14,15-EET-induced inhibition was potentiated by 4-phenylchalcone oxide, an epoxide hydrolase inhibitor. The inhibition occurred when substrate amounts of AA were used and was not accompanied by enhanced production of other PGs, suggesting an effect on PGH synthase; however, in murine cells, 14,15-EET did not reduce PGH synthase mRNA or protein. Moreover, the 14,15-EET-induced decrease in PGE₂production was overcome by increasing the concentration of AA, but not oleic acid (which is not a substrate for PGH synthase). These findings suggest that 14,15-EET competitively inhibits PGH synthase activity in vascular smooth muscle cells. The 14,15-EET-induced inhibition of PGE₂production resulted in potentiation of platelet-derived growth factor-induced smooth muscle cell proliferation, suggesting that the competitive inhibition of PGH synthase by 14,15-EET can affect growth responses in smooth muscle cells.