The Cytochrome P450 (CYP450) metabolite 20-HETE plays a significant role in pressure-induced vasoconstriction (myogenic response) in resistance arteries. It is less clear whether this role is due to regulation of stretch-activated ion channels involved in the myogenic response. Transient receptor potential (TRP) channels are expressed in mammalian vascular smooth muscle cells (VSMC) and have been suggested as the stretch sensor involved in myogenic responsiveness. We investigated the possibility that specific inhibition of 20-HETE production interferes with stretch-induced activation of functionally expressed TRPC6 channels. To clarify the physiological relevance of such a mechanism we examined the effect of specific inhibition of 20-HETE production on myogenic tone in rat mesenteric arteries (RMA). Freshly excised RMA (2nd or 3rd order) obtained from humanely killed animals were mounted in a pressure myograph and superfused with HCO₃^–/CO₂-buffered PSS (pH 7.4) at 37°C. Steady-state diameter was recorded at intraluminal pressures of 0, 20, 40, 60, 80 and 100 mmHg during control and drug periods. The passive pressure-diameter relationship was obtained in Ca²⁺-free solution (+EGTA). Whole-cell currents and Ba²⁺ fluorescence were measured on HEK293 cells transiently expressing TRPC6 channels by using the patch-clamp and fluorescence imaging techniques, and stretch-activation was induced by hypotonic solution (-90 mOsm). Specific inhibition of 20-HETE production was obtained by using HET0016 (Taisho Pharmaceutical Co., Saitama, Japan), a selective blocker of CYP450 4A enzyme catalysing 20-HETE production, and unspecific inhibition was obtained by using 17-ODYA. RMA did not develop substantial myogenic tone under control conditions. To mimic sympathetic nerve activity, RMA were preconstricted with low phenylephrine concentration (0.05—1.0 μM) to ∼85% of the resting diameter at 60 mmHg. Preconstriction resulted in decreased diameter at pressures ≥20 mmHg as compared with the passive curve (P<0.05, N=4). However, in separate RMA (N=4) with similar degree of preconstriction, pretreatment with 10 μM HET0016 eliminated this difference. In TRPC6–expressing HEK293 cells, hypotonic challenge potentiated both receptor-activated (100 μM carbachol) cation current and Ba²⁺ entry about 2-fold (P<0.01, n=8), and this effect was abolished by pretreatment with HET0016 (3—10 μM) or 17-ODYA (10 μM). Hypotonic potentiation of cation current or Ba²⁺ entry was not detectable in the absence of receptor stimulation. Our data shows that 20-HETE production is important for development of myogenic tone induced by low level of α-adrenoceptor stimulation in RMA. The mechanism responsible may be 20-HETE mediated potentiation of receptor-activated TRPC6 channels in VSMC as was demonstrated in HEK293 cells.