Objectives: Peroxynitrite (ONOO⁻) mediates in part both ischemia–reperfusion and pro-inflammatory cytokine-induced injury to the heart. As oxidants like ONOO⁻ are known to activate matrix metalloproteinases (MMPs), we examined whether they play a role in the detrimental action of ONOO⁻ in isolated perfused rat hearts. Methods: Hearts were isolated from Sprague–Dawley rats and perfused retrogradely with Krebs–Henseleit buffer under constant flow. Peroxynitrite (30 and 80 μM) was infused into the hearts for 15 min. The release of MMPs into the coronary effluent and level of MMPs in the myocardium were measured by gelatin zymography. Results: The main gelatinolytic activity in control effluent was 72-kDa corresponding to pro-MMP-2. Infusion of ONOO⁻ (80 μM) for 15 min caused a vasodilatation which peaked at 5 min and then converted into vasoconstriction by 15 min. It also caused a rapid increase in the release of 72-kDa activity within 10 min and a progressive decline in cardiac mechanical function. In contrast, decomposed ONOO⁻ caused no change in vascular tone, the release of 72-kDa activity or mechanical function. The MMPs inhibitor PD-166793 prevented the ONOO⁻-induced loss in myocardial mechanical function. Detoxification of ONOO⁻ with glutathione prevented both the enhancement in coronary effluent 72-kDa activity and the decline in mechanical function. Conclusions: Acute cardiac toxicity induced by ONOO⁻ is mediated by MMP-2.