The main objective of this work was to study the role of potassium channels in the cholinergic relaxation of cutaneous arteries during cooling. Acetylcholine (10<sup>–8</sup>–10<sup>–4</sup> M) produced isometric concentration-dependent relaxation of precontracted segments of rabbit ear (cutaneous) and femoral (noncutaneous) arteries; this relaxation was higher at 24 ° C (cooling) than at 37 °C in ear, but not in femoral, arteries. In both types of arteries, at 37 and 24 °C, the relaxation to acetylcholine was partially reduced by the inhibitor of nitric oxide synthase N<sup>G</sup>-nitro- L-arginine methyl ester ( L-NAME, 10<sup>–4</sup> M), and the relaxation that remained after L-NAME was higher at 24°C than at 37°C in ear, but not in femoral, arteries. At 37 and 24°C, the persistent relaxation to acetylcholine after L-NAME was further reduced by smooth muscle depolarization with medium containing a high concentration of potassium (6 × 10<sup>–2</sup> M), and with the nonspecific inhibitors of potassium channels tetraethylammonium (10<sup>–2</sup> M) or 4-aminopyridine (5 × 10<sup>–3</sup> M) in both ear and femoral arteries. In ear arteries, the inhibitor of high conductance calcium-activated potassium channels charybdotoxin (10<sup>–7</sup> M), alone or combined with L-NAME, reduced the relaxation to acetylcholine at 24°C, but not at 37°C. In femoral arteries, charybdotoxin alone did not modify, but combined with L-NAME reduced, the relaxation to acetylcholine at either temperature. At 37 and 24 °C, the inhibitor of low conductance calcium-activated potassium channels apamin (10<sup>–7</sup> M), the inhibitor of ATP-dependent potassium channels glibenclamide (10<sup>–5</sup> M) and the cyclooxygenase inhibitor meclofenamate (10<sup>-5</sup> M), alone or combined with L-NAME, did not modify the relaxation of both ear and femoral arteries to acetylcholine. These results suggest: (1) the cholinergic relaxation of cutaneous (ear) and noncutaneous (femoral) arteries could be mediated by endothelial nitric oxide and by activation of potassium channels, and (2) cooling increases the relaxation of cutaneous arteries to cholinergic stimulation, which may be mediated, in part, by an increased response of potassium channels.