Carbon monoxide (CO) plays an important physiological role in regulation of the vascular tone. CORM-2 is frequently used as a CO-donor to evaluate the physiological and pathophysiological properties of CO as well as the potential therapeutic applications of this diatomic molecule. The aim of this study was to examine the molecular mechanisms underlying the vasodilatory properties of CORM-2 as this has not yet been extensively explored. Isometric tension recordings were performed using different isolated blood vessels from both mice and rats. Responses to saturated CO solutions and CORM-2 were evaluated in the presence/absence of activators/inhibitors of different molecular pathways. The saturated CO solution was unable to relax mice isolated blood vessels, whereas it induced concentration-dependent relaxations in rat aortic ring segments. The response to CO was inhibited by both the soluble guanylyl cyclase inhibitor ODQ and potassium channel blocker TEA. CORM-2 relaxed both mice and rat isolated blood vessels in a concentration-dependent manner. The vasodilatory response was however only partially blocked by ODQ and TEA. Interestingly, 4-aminopyridine antagonised the CORM-2 induced vasodilatation whereas iberiotoxin had no influence on the response elicited by this CO-releasing molecule. It is concluded that the molecular mechanisms involved in CORM-2 induced vasodilatation differ from the mechanisms underlying CO induced vasorelaxation. CO induces vasorelaxation by activating sGC and/or calcium-activated potassium channels. In contrast, the vasodilatory properties of CORM-2 are only partially dependent upon sGC or potassium channel activation. Moreover, the CORM-2 induced vasodilatation seems to involve voltage-dependent potassium channels instead of calcium-activated potassium channels.