Cardiomyocyte hypertrophy is associated with multiple pathophysiological cardiovascular conditions. Recent studies have substantiated the finding that oxidants may contribute to the development of cardiomyocyte hypertrophy. Activation of NFAT3 transcription factor has been shown to result from endocrine inducers of cardiomyocyte hypertrophy such as angiotensin II (AngII) and serves as an important molecular regulator of cardiomyocyte hypertrophy. In this study, we found that antioxidant enzyme catalase and antioxidants N-acetyl-L-cysteine, -phenyl-n-tert-butylnitrone (PBN) and lipoic acid prevent AngII from activating NFAT3 promoter-luciferase. H₂O₂ induces a time- and dose-dependent activation of NFAT3 transcription factor. A dominant negative form of NFAT3 transcription factor inhibited H₂O₂ from activating NFAT3 promoter. An inhibitor of ERKs, but not PI3K or p38 MAPKs, blocked NFAT3 activation by H₂O₂ . The NFAT3 binding site in the promoters of most genes contains a weak AP-1 binding site adjacent to the core consensus NFAT binding sequence. ERKs inhibitor PD98059 was found previously to inhibit AP-1 activation by H₂O₂ . Inactivating AP-1 transcription factor by cotransfecting a dominant negative c-Jun, TAM67, prevented H₂O₂ or AngII from activating NFAT3 promoter. NFAT3 promoter containing the core NFAT cis-element without AP-1 binding site failed to show activation by H₂O₂ treatment. Our data suggest that hypertrophy inducers AngII and H₂O₂ may activate NFAT3 in cardiomyocyte through an AP-1 transcription factor dependent mechanism.