[Arg⁸]-vasopressin (AVP), at low concentrations (10-500 pM), stimulates oscillations in [Ca²⁺]_i (Ca²⁺ spikes) in A7r5 rat aortic smooth muscle cells. Our previous studies provided biochemical evidence that protein kinase C (PKC) activation and phosphorylation of voltage-sensitive K⁺ (K_v) channels are crucial steps in this process. In the present study, K_v currents (I_Kv) and membrane potential were measured using patch clamp techniques. Treatment of A7r5 cells with 100 pM AVP resulted in significant inhibition of I_Kv. This effect was associated with gradual membrane depolarization, increased membrane resistance, and action potential (AP) generation in the same cells. The AVP-sensitive I_Kv was resistant to 4-aminopyridine, iberiotoxin, and glibenclamide, but was fully inhibited by the selective KCNQ channel blockers, linopirdine (10 µM) and XE991 (10 µM) and enhanced by the KCNQ channel activator, flupirtine (10 µM). BaCl₂ (100 µM) or linopirdine (5 µM) mimicked the effects of AVP on K⁺ currents, AP generation, and Ca²⁺ spiking. Expression of KCNQ5 was detected by RT-PCR in A7r5 cells and freshly isolated rat aortic smooth muscle. RNA interference directed toward KCNQ5 reduced KCNQ5 protein expression and resulted in a significant decrease in I_Kv in A7r5 cells. I_Kv was also inhibited in response to the PKC activator, 4-phorbol 12-myristate 13-acetate (10 nM), and the inhibition of I_Kv by AVP was prevented by the PKC inhibitor, calphostin C (250 nM). These results suggest that the stimulation of Ca²⁺ spiking by physiological concentrations of AVP involves PKC-dependent inhibition of KCNQ5 channels and increased action potential firing in A7r5 cells.