The mechanisms of metallothionein prevention of diabetic cardiomyopathy are largely unknown. The present study was performed to test whether inhibition of nitrosative damage is involved in metallothionein prevention of diabetic cardiomyopathy. Cardiac-specific metallothionein-overexpressing transgenic (MT-TG) mice and wild-type littermate controls were treated with streptozotocin (STZ) by a single intraperitoneal injection, and both developed diabetes. However, the development of diabetic cardiomyopathy, revealed by histopathological and ultrastructural examination, serum creatine phosphokinase, and cardiac hemodynamic analysis, was significantly observed only in the wild-type, but not in MT-TG, diabetic mice 2 weeks and 6 months after STZ treatment. Formations of superoxide and 3-nitrotyrosine (3-NT), a marker for peroxynitrite-induced protein damage, were detected only in the heart of wild-type diabetic mice. Furthermore, primary cultures of cardiomyocytes from wild-type and MT-TG mice were exposed to lipopolysaccharide/tumor necrosis factor-alpha for generating intracellular peroxynitrite. Increases in 3-NT formation and cytotoxicity were observed in wild-type, but not in MT-TG, cardiomyocytes. Either urate, a peroxynitrite-specific scavenger, or Mn(111) tetrakis 1-methyl 4-pyridyl porphyrin pentachloride (MnTMPyP), a superoxide dismutase mimic, significantly inhibited the formation of 3-NT along with a significant prevention of cytotoxicity. These results thus suggest that metallothionein prevention of diabetic cardiomyopathy is mediated, at least in part, by suppression of superoxide generation and associated nitrosative damage.