Ion channel inhibitors are widely used for pharmacological discrimination between the different channel types as well as for determination of their functional role. In the present study, we tested the hypothesis that 4-aminopyridine (4-AP) could affect the large conductance Ca2⁢-activated K⁢ channel (BKCa) currents using perforated-patch or cell-attached configuration of patch-clamp technique in the rabbit pulmonary arterial smooth muscle. Application of 4-AP reversibly inhibited the spontaneous transient outward currents (STOCs). The reversal potential and the sensitivity to charybdotoxin indicated that the STOCs were due to the activation of BKCa. The BKCa currents were recorded in single channel resolution under the cell-attached mode of patch-clamp technique for minimal perturbation of intracellular environment. Application of 4-AP also inhibited the single BKCa currents reversibly and dose-dependently. The membrane potential of rabbit pulmonary arterial smooth muscle cells showed spontaneous transient hyperpolarizations (STHPs), presumably due to the STOC activities, which was also inhibited by 4-AP. These results suggest that 4-AP can inhibit BKCa currents in the intact rabbit vascular smooth muscle. The use of 4-AP as a selective voltage-dependent K⁢ (KV) channel blocker in vascular smooth muscle, therefore, must be reevaluated.