Voltage-gated K⁺ (K_V ) channels are the predominant K⁺ channels expressed in pulmonary vasculature and have been thought to play an important role in regulation of vessel excitability and contribute to hypoxic pulmonary vasoconstriction (Archer & Michelakis, 2002). Although K_V channel currents were well characterised in isolated pulmonary arterial myocytes, their relative contribution to contraction of intact pulmonary arteries remains incompletely understood. The main aim of this work was to investigate the contribution of the K_V channels in small intrapulmonary arteries (PAs) isolated from male Wister rats (200-250 gm) using small vessel wire myography and a specific inhibitor of K_V channels 4-aminopyridine (4-AP). Small mesenteric arteries (MAs) were used as a representative of the systemic circulation. In PAs, application of 4-AP (0.5-10 mM) caused contraction typically only at 5 or 10 mM. Membrane depolarisation with 20 mM KCl significantly potentiated (p < 0.001, t-test) the effect of 4-AP, probably by causing greater activation of the K_V channels. Pre-treatment of PAs with the selective inhibitor of background KCNQ channels linopirdine (10 μM) similarly significantly potentiated (p < 0.004, t-test) concentration-dependent 4-AP induced contraction causing a marked contraction even at 0.5 mM 4-AP, the concentration which block ~50% of K_V currents in single cells (Smirnov et al. 2000). Potentiation of 4-AP-induced contraction by linopirdine and 20 mM KCl was significantly less in MAs. These results suggest that K_V and KCNQ channels are important regulators of the resting cell membrane potential in PAs. 10 mM 4-AP-induced contraction of PAs developed in the presence of 10 μM linopirdine was only partly blocked by the inhibitor of L-type Ca²⁺ channels diltiazem (10 µM, 35±4%, n=6) or by the selective Rho kinase inhibitor Y-27632 (10 μM, 37±14%, n=6). In MAs, the effects of diltiazem (94±2%, n=6) and Y-27632 (66±12%, n=6) on the 4-AP-induced contraction recorded under identical condition was significantly greater than in PAs (p < 0.001, t-test). The effects of Y-27632 is likely to reflect the inhibition of the basal level of the enzyme activity, because the pre-treatment of arteries with 4-AP (10 mM) did not change levels of phosphorylated regulatory myosin phosphatase target subunit (MYPT-1). In alpha-toxin permeabilized tissues, pre-treatment with 3 μM carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), a mitochondrial uncoupler, significantly reduced 4-AP contraction in PAs and not in MAs. In conclusion, our results demonstrate the involvement of at least two types of K⁺ channels, the K_V and KCNQ channels, in the regulation of PA contractility and also provide indirect evidence for possible involvement of mitochondria in contraction mediated by inhibition of K_V channels.