Kir6.1/SUR2B channel is the major isoform of K_ATP channels in the vascular smooth muscle. Genetic disruption of either subunit leads to dysregulation of vascular tone and regional blood flows. To test the hypothesis that the Kir6.1/SUR2B channel is a target molecule of arginine vasopressin (AVP), we performed the studies on the cloned Kir6.1/SUR2B channel and cell-endogenous K_ATP channel in rat mesenteric arteries. The Kir6.1/SUR2B channel was expressed together with V1a receptor in the HEK293 cell line. Whole-cell currents of the transfected HEK cells were activated by K_ATP channel opener pinacidil and inhibited by K_ATP channel inhibitor glibenclamide. AVP produced a concentration-dependent inhibition of the pinacidil-activated currents with IC₅₀ 2.0 nM. The current inhibition was mediated by a suppression of the open-state probability without effect on single channel conductance. An exposure to 100 nM PMA, a potent PKC activator, inhibited the pinacidil-activated currents, and abolished the channel inhibition by AVP. Such an effect was not seen with inactive phorbol ester. A pretreatment of the cells with selective PKC blocker significantly diminished the inhibitory effect of AVP. In acutely dissociated vascular smooth myocytes, AVP strongly inhibited the cell-endogenous KATP channel. In isolated mesenteric artery rings, AVP produced concentration-dependent vasoconstrictions with EC₅₀ 6.5nM. At the maximum effect, pinacidil completely relaxed the vasoconstriction in the continuing exposure to AVP. The magnitude of the AVP-induced vasoconstriction was significantly reduced by calphostin-C. These results therefore indicate that the Kir6.1/SUR2B channel is a target molecule of AVP, and the channel inhibition involves G_q-coupled V1a receptor and PKC.