We have previously shown that the inhibition of myocardial nitric oxide (NO) and peroxynitrite-matrix metalloproteinase (MMP) signaling by early preconditioning (PC) is involved in its cardioprotective effect. Therefore, in the present study, we investigated the role of NO and peroxynitrite-MMP signaling in the development of late PC. PC was performed by five consecutive cycles of 4-min coronary occlusion and 4-min reperfusion in anesthetized rats in vivo. Twenty-four hours later, hearts were subjected to a 30-min coronary occlusion followed by 180-min reperfusion to measure infarct size. In separate experiments, heart tissue was sampled to measure biochemical parameters before and 3, 6, 12, or 24 h after the PC protocol, respectively. Late PC decreased infarct size, increased cardiac inducible NO synthase (iNOS) activity and gene expression, and decreased SOD activity at 24 h significantly compared with sham-operated controls. Late PC increased cardiac superoxide levels significantly at 24 h; however, it did not change cardiac NO levels. Cardiac peroxynitrite levels were significantly decreased. Downstream cellular targets of peroxynitrite, MMP-2 and MMP-9 activities were decreased in the late PC group at 24 h compared with the sham-operated group. To verify if PC-induced inhibition of MMPs had a causative role in the reduction of infarct size, in separate experiments, we measured infarct size after the pharmacological inhibition of MMPs by ilomastat and found a significant reduction of infarct size compared with the vehicle-treated group. In conclusion, this is the first demonstration that the inhibition of cardiac peroxynitrite-MMP signaling contributes to cardioprotection by late PC and that pharmacological inhibition of MMPs is able to reduce infarct size in vivo. Furthermore, increased expression of iNOS may play a role in the development of late PC; however, increased iNOS activity does not lead to increased NO production in late PC.