HETE, a cytochrome P-450 4A (CYP4A1)-derived arachidonic acid metabolite, is a major eicosanoid formed in renal and extrarenal microcirculation. 20-HETE inhibits Ca 2ϩ -activated K ϩ channels in vascular smooth muscle cells and thereby may modulate vascular reactivity. We transfected renal interlobar arteries with an expression plasmid containing the cDNA of CYP4A1, the low-Km arachidonic acid -hydroxylase, and examined the consequences of increasing 20-HETE synthesis on constrictor responses to phenylephrine. CYP4A1-transfected interlobar arteries demonstrated a twofold increase in CYP4A protein levels and 20-HETE production compared with arteries transfected with the empty plasmid; they also showed increased sensitivity to phenylephrine, as evidenced by a decrease in EC50 from 0.37 Ϯ 0.04 M in plasmid-transfected arteries to 0.07 Ϯ 0.01 M in CYP4A1-transfected arteries. The increased sensitivity to phenylephrine was greatly attenuated by , a selective inhibitor of 20-HETE synthesis, and by 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid, a specific 20-HETE antagonist. This effect of DDMS was reversed by addition of 20-HETE, further substantiating the notion that increased levels of 20-HETE contribute to the increased sensitivity to phenylephrine in vessels overexpressing CYP4A1. These data suggest that 20-HETE of vascular origin sensitizes renal vascular smooth muscle to phenylephrine. 20-hydroxyeicosatetraenoic acid; arachidonic acid; phenylephrine; cytochrome P-450 SMALL ARTERIAL VESSELS MANUFACTURE 20-HETE, a product of arachidonic acid metabolism by cytochrome P-450 enzymes of the 4A family (CYP4A1) (8,12,17,26). Exogenous 20-HETE was reported to contract or relax vascular smooth muscle, depending on the animal species, type of vessels, and experimental conditions. Cyclooxygenase-dependent and -independent mechanisms have been implicated in 20-HETE-induced vascular contraction (6, 16, 17) and vascular relaxation (2, 4).Renal preglomerular vessels express CYP4A proteins, produce 20-HETE, and are constricted by exogenous 20-HETE. The constrictor action of exogenous 20-HETE in pressurized canine arcuate arteries, rat renal interlobular arteries, and rat renal afferent arterioles is independent of cyclooxygenase activity and has been attributed to inhibition of the opening of Ca 2ϩ -activated K ϩ channels in vascular smooth muscle cells, leading to cellular depolarization and increased Ca 2ϩ entry (8,12,14,23,28). Recent studies support the notion that endogenous 20-HETE subserves vasconstrictor mechanisms in the rat kidney. For example, inhibitors of 20-HETE synthesis were shown to increase blood flow to the kidney (29), to increase the diameter of preconstricted interlobular arteries denuded of endothelium (11), and to attenuate vasconstrictor responses elicited by endothelin (10, 22), angiotensin II (5), or increases in transmural pressure (14). 20-HETE produced by the smooth muscle isolated from renal preglomerular vessels was also shown to exert a tonic inhibitory influence on the activity of large-conductance C...