The voltage-gated K+ channel Kv1.5 has key roles in the regulation of the vascular system1 and in atrial excitability2, and contributes to apoptosis in various tissues. In this study we examined the regulation of recombinant and native Kv1.5 by carbon monoxide (CO); a product of the cytoprotective heme oxygenase enzymes, with recognized beneficial cardiovascular effects3. Whole cell patch clamp recordings were made from HEK293 cells expressing recombinant hKv1.5. Values are means ± S.E.M. Bath application of CO was achieved using the CO donor molecule CORM-2, which reversibly inhibited Kv1.5 in a concentration-dependent manner by 55.2 ±3.78%, n=19 measured at 50mV (30µM; P< 0.05, Student’s paired t test). Application of the inactive form (iCORM) had no significant effect. To determine the mechanism behind this CO effect we examined multiple signalling pathways; the actions of CO were significantly suppressed by the reducing agent DTT (1mM) and the superoxide dismutase (SOD) mimetics MnTMPyP (50µM) or MnTBAP (10µM) (85.8 ±1.14%, n=5; 79.2 ±0.85%, n=5; 81.1 ±6.4%, n=5, P< 0.05 respectively) indicating an involvement of reactive oxygen species (ROS). Moreover, pretreatment of cells with the sGC inhibitor, Rp-8-Br-cGMPS (100nM) or the NOS inhibitor, L-NAME (1mM, 1hr at 37°C) significantly reduced the inhibitory effects of CORM-2 on Kv1.5 by 73.8 ±1.9%, n=5 and 81.8 ±7.45%, n=6, P< 0.05, indicating involvement of NO formation. CO also elevated intracellular peroxynitrite (ONOO-) formation, and a peroxynitrite scavenger FeTPPS markedly attenuated the ability of CO to inhibit Kv1.5 by 79.9 ±5.7, n=5, P< 0.05, measured via the ONOO− indicator, 2-[6-(4′-amino) phenoxy-3H-xanthen-3-on-9-yl]benzoic acid (APF; n=5). CO also caused nitrosylation of Kv1.5, and two mutant forms of the channel, C331A and C346A (n=3). Hydrogen peroxide exposure induced augmentation of Kv1.5 by 15.62 ± 4.4% (n=4; P< 0.05); an effect fully reversed by CO. In murine atrial HL-1 cells CO inhibited native Kv1.5/Ikur by 49.84 ±4.9% (n=10 measured at 50mV), prolonged spontaneous action potentials and increased their amplitude by 7.2 ±1.5 mV, n=6. A similar effect was observed when HL-1 cells were exposed to DPO-1, a blocker of KV1.5 channel and the IKur current. Furthermore, in the presence of DPO-1, CO did not increase the amplitude or duration of spontaneous action potentials further (n=6), suggesting that both CO and DPO-1 may act at the same site. Our data indicate that Kv1.5 is a target for modulation by CO via multiple mechanisms4. This regulation has important implications for its diverse cellular functions, including excitability, contractility and apoptosis.