Background. The AMP-activated protein kinase (AMPK) is intimately coupled to mitochondrial metabolism through changes in the AM(D)P:ATP ratios induced by metabolic stresses such as hypoxia [1, 2]. Of the various known Kv channel types it has been established that Kv1.5 contributes the majority of the macroscopic voltage-gated potassium currents recorded from myocytes of near resistance-sized pulmonary arteries, that exhibit the greatest response to hypoxia [3]. Kv current suppression during hypoxia [3] occurs as a consequence of inhibition of mitochondrial oxidative phosphorylation [4] and mediates, in part, progression of hypoxic pulmonary hypertension [3]. However, the nature of the signalling pathway that couple mitochondrial function to Kv channels has been unclear. We sought to determine the role of AMPK in this process. Methods. Myocytes were enzymatically isolated from resistance pulmonary arteries (<200 µm i.d.) of male Sprague Dawley rats. Kv currents were recorded using whole-cell patch clamp in voltage-clamp mode with a holding potential of -80 mV. Kv currents were assessed by voltage ramps (-100 to +40 mV), single voltage steps (-80 to +40 mV), and by acquisition of full current-voltage relationships for steady state activation (200 ms steps from -80 mV to +40 mV in 10 mV increments). Isoform-specific AMPK activities were determined by immunoprecipitating tissue lysate with antibodies raised against α1 or α2 subunits bound to protein G-Sepharose beads and quantified using the AMARA peptide and [γ-32P]ATP substrates. Phosphorylation assays were performed as described previously [5]. Results. Inhibition of the mitochondrial electron transport chain using phenformin activated AMPK and inhibited Kv currents in pulmonary arterial myocytes, consistent with previously reported effects of mitochondrial inhibitors [4]. Myocyte Kv currents were also markedly inhibited by application of three AMPK activators with distinct mechanisms of action, i.e., A769662, AICAR and C13. Hypoxia and inhibitors of mitochondrial oxidative phosphorylation (phenformin and antimycin A) reduced Kv1.5 currents and blocked further inhibition by AMPK activators, as did the selective Kv1.5 blocker DPO-1. Moreover, recombinant human Kv1.5 channels were phosphorylated by AMPK in cell-free assays, suggesting direct regulation of the channel by AMPK. Conclusion. These results suggest that AMPK is the primary regulator of reductions in Kv1.5 channel availability in pulmonary arterial myocytes following inhibition of mitochondrial oxidative phosphorylation during hypoxia, and that AMPK effects this change, at least in part, through phosphorylation of Kv1.5 and/or an associated protein.