The research studied the concentration variation of peroxynitrite anion (O=N-O-O-) released from cultured neonatal myocardial cells induced by ischemia/reperfusion and studied the protective effect of melatonin on the injury. For this purpose, amperometry peroxynitrite ultramicrosensors (UMS) were fabricated and constructed by electropolymerizing inorganic macromolecular film of tetraaminophthalocyanine manganese(II) and coating chemically with poly(4-vinylpyridine). Under optimum conditions, the UMS showed high selectivity and sensitivity to peroxynitrite determination with a calculated detection limit of 1.8 x 10(-8) mol/L (S/N of 3). The detection of peroxynitrite was based on electrocatalytic reduction of peroxynitrite. The mechanism of catalysis was also discussed. The UMS should be promising for in vivo measurement of peroxynitrite without interference or fouling. Peroxynitrite released from myocardial cells both in the ischemic period and in the reperfusion period was measured directly. This approach may lead to important information for myocardial cells on the mechanism of injury and prospective treatments of medicine such as melatonin.