Carbon monoxide (CO) is believed to mediate many of the cytoprotective effects attributed to the activation of heme oxygenase (HO-1), the enzyme responsible for CO production. Recently, the study of CO-releasing molecules (CO-RMs) has provided a new approach for the delivery of CO. In the present study, we examined whether the cardioprotective properties of CO-RM2 in isolated rat hearts subjected to an ischemia-reperfusion (I/R) sequence were associated with the presence of CO. In addition, the antioxidant properties of CO-RM2 were evaluated. In hearts pretreated with CO-RM2, the improvement in contractile function at the end of the reperfusion period after 20 min of global total ischemia was significantly greater than in controls. These beneficial effects were accompanied by a reduction in 1) LDH activity release 2) infarct size 3) ventricular superoxide production. The improvement in myocardial function and the reduction in oxidative stress were not observed when hearts were pretreated with inactivated CO-RM2 (iCO-RM2). Additionally, CO-RM2, but not iCO-RM2, was found to exert antioxidant properties. These results suggest that the production of CO is a necessary factor in the cardioprotective and antioxidant actions of CO-releasing compound. These results may open up new ground for a novel class of cardioprotective compounds.