The present study examined whether 20-hydroxyeicosatetraenoic acid (HETE) contributes to the vasoconstrictor effect of angiotensin II (ANG II) in renal microvessels by preventing activation of the large conductance Ca 2+-activated K + channel (K Ca) in vascular smooth muscle (VSM) cells. ANG II increased the production of 20-HETE in rat renal microvessels. This response was attenuated by the 20-HETE synthesis inhibitors, 17-ODYA and HET0016, a phospholipase A 2 inhibitor AACOF 3, and the AT 1 receptor blocker, Losartan, but not by the AT 2 receptor blocker, PD123319. ANG II (10 -11 to 10 -6 M) dose-dependently decreased the diameter of renal microvessels by 41 ± 5%. This effect was blocked by 17-ODYA. ANG II (10 -7 M) did not alter K Ca channel activity recorded from cell-attached patches on renal VSM cells under control conditions. However, it did reduce the NPo of the K Ca channel by 93.4 ± 3.1% after the channels were activated by increasing intracellular calcium levels with ionomycin. The inhibitory effect of ANG II on K Ca channel activity in the presence of ionomycin was attenuated by 17-ODYA, AACOF 3, and the phospholipase C (PLC) inhibitor U-73122. ANG II induced a peak followed by a steady-state increase in intracellular calcium concentration in renal VSM cells. 17-ODYA (10 -5 M) had no effect on the peak response, but it blocked the steady-state increase. These results indicate that ANG II stimulates the formation of 20-HETE in rat renal microvessels via the AT 1 receptor activation and that 20-HETE contributes to the vasoconstrictor response to ANG II by blocking activation of K Ca channel and facilitating calcium entry.