The purpose of this study is to investigate whether an oxygen/ozone (O(2)/O(3)) mixture protects the heart from acute myocardial infarction through local involvement of endothelial nitric oxide synthase (eNOS) and endothelial progenitor cells (EPCs). Male Sprague-Dawley rats were subject to 25-min occlusion and 2-h reperfusion of the left descending coronary artery. O(2)/O(3) mixture was insufflated i.p. 30 min prior to ischemia/reperfusion (I/R) procedure at doses of 100, 150, and 300 microg/kg. Myocardial infarct size (IS) measurement and myocardial immunohistochemistry for EPCs were done. For these latter cells, immunoreactivities for CD34, and CD117/c-kit were assessed within the infarcted tissue. Moreover, cardiac eNOS was monitored. I/R in rats treated with O(2) produced an IS as a percentage of the area at risk (IS/AR) equal to 51 +/- 5%. I/R in rats treated with the O(2)/O(3) mixture showed reduced IS (for example, IS/AR for 150 microg/kg O(2)/O(3) was 35 +/- 2.1%; P < 0.01 vs. O(2)). The O(2)/O(3) cardio-protection was paralleled by an increased number of immunopositive particles per area for CD34 and CD117/c-kit. The increase of these markers was associated with an increase of cardiac eNOS expression as assayed by immunohistochemistry. Interestingly, N5-(1-Iminoethyl)-L-ornithine dihydrochloride (L-NIO), a selective eNOS activity inhibitor (30 mg/kg s.c.), prior to O(2)/O(3) and I/R almost abolished the cardio-protection exerted by the O(2)/O(3) mixture. L-NIO also abolished the increase in immunostaining for CD-34 and CD117/c-kit as compared with the rats receiving O(2)/O(3) and I/R only. O(2)/O(3) mixture protects the heart from acute myocardial infarction through local increase of eNOS expression/activity and consequent EPCs recruitment.