In vivo administration of L-arginine at different time points during the course of myocardial ischemia and reperfusion (MI/R) has been shown to differentially regulate postischemic apoptosis. Cardiac function is one of the most important indexes used to judge the degree of myocardial injury. The present study attempted to determine whether in vivo administration of L-arginine at different stages of MI/R has a diverse influence on cardiac function of ischemic reperfused hearts and, if so, to investigate the mechanisms involved. Male adult rats were subjected to 30 min myocardial ischemia followed by 5 h reperfusion. An intravenous L-arginine bolus was given either 10 min before and 50 min after reperfusion (early treatment) or 3 h and 4 h after reperfusion (late treatment). Early treatment with L-arginine markedly increased the left ventricular systolic pressure (LVSP) and dP/dtmax, and decreased myocardial nitrotyrosine content. In strict contrast, late treatment with L-arginine resulted in a significant decrease in LVSP and dP/dtmax from 4 h to 5 h after reperfusion, and increase in toxic peroxynitrite formation as measured by nitrotyrosine. These results suggest that the administration of L-arginine at different time points during the course of MI/R leads to diverse effects on cardiac dysfunction. Early supplementation decreased the nitrative stress and improved left ventricular function. However, late treatment with L-arginine increased the formation of peroxynitrite and aggravated cardiac functional injury.