The effect of myocardial ischemia on nitric oxide (NO) production is controversial in part because of indirect NO quantification. In the present study, direct quantification of NO was investigated in an in vivo rat model of myocardial ischemia (MI). A NO spin-trapping technique using electron spin resonance (ESR) spectroscopy was used to study NO production in the ischemic and in the nonischemic area of the rat heart 2, 8, or 24 h after left main coronary artery ligation. The method was based on the trapping of NO by a metal-chelator complex consisting of N-methyl-D-glucamine-dithiocarbamate (MGD) and Fe (II) to form a stable NO-FeMGD complex that gives rise to a characteristic triplet ESR spectrum. This metal-chelator complex was administered half an hour before sacrifice of the rats. A large and time-dependent increase of the ESR signal corresponding to the NO-FeMGD complex was observed 8 h (11.6±0.9 arbitrary units [AU]) and 24 h (29.7±2.9 AU) in the ischemic area after MI. On the contrary, no ESR triplet was observed in the nonischemic region of the heart and in sham-operated rats. NO blood derivative levels (nitrosylhemoglobin and plasma nitrites and nitrates) were unchanged compared with sham-operated rats. Previous administration of aminoguanidine, a NO synthase inhibitor, in animals subjected to a 24-h ischemia resulted in a complete abolition in the NO-FeMGD spectrum in the ischemic area. These findings directly demonstrated an increase of the NO-FeMGD levels during in vivo myocardial ischemia that appeared to be specifically localized in the ischemic area.