Voltage-dependent K+ (Kv) channels were studied in smooth muscle cells (SMCs) freshly isolated from rat mesenteric arteries. A delayed outward rectifier Kv current (IK) with a weak voltage dependence was identified. The amplitude of IK, but not its inactivation kinetics, was inhibited by 4-aminopyridine (4-AP) (IC50, 5.1 +/- 0.9 mM). The inhibitory effect of 4-AP was not use-dependent, and the unbinding of 4-AP from IK channels was complete in the absence of depolarization stimuli, suggesting the binding of 4-AP to the closed state of Kv channels. There was no change in the steady-state inactivation, but the steady-state activation curve of IK was shifted in the presence of 4-AP by +6 mV. Including 4-AP in pipette solution instantly inhibited IK upon the rupture of cell membrane, indicating that 4-AP bound to the inner mouth of Kv channel pores. Several Kv channel proteins encoding the native IK-type Kv channels, but not the transient outward A-type Kv channels, were identified. Among the identified IK-encoding gene transcripts, the expression of Kv1.5 was the most abundant. Our results elucidate the modulating mechanisms for the 4-AP-induced IK inhibition in rat mesenteric artery SMCs and suggest that the unique properties of Kv channels in these cells might be related to the heteromeric expression of the IK-encoding genes with Kv1.5 subunit playing an important role.