The role of nitric oxide (NO) generated by the inducible NO synthase (iNOS) during myocardial ischemia and reperfusion is not understood. We investigated the role of iNOS during early reperfusion damage induced in genetically deficient iNOS (iNOS^‐/‐) mice and wild‐type littermates. In wild‐type mice, ischemia (60 min) and reperfusion (60 min) induced an elevation in serum levels of creatine phosphokinase and myocardial injury characterized by the presence of scattered apoptotic myocytes and mild neutrophil infiltration. Northern blot analysis showed increased expression of iNOS, whose activity was markedly elevated after reperfusion. Immunohistochemistry showed staining for nitrotyrosine; Western blot analysis showed elevated expression of heat shock protein 70 (HSP70), a putative cardioprotective mediator. Plasma levels of nitrite and nitrate, tumor necrosis factor α (TNF‐α), interleukin 6 (IL‐6), and IL‐10 were also increased. These events were preceded by degradation of inhibitor κBα (IκBα), activation of IκB kinase complex (IKK) and c‐Jun‐NH₂‐terminal kinase (JNK), and subsequently activation of nuclear factor‐κB (NF‐κB) and activator protein 1 (AP‐1) as early as 15 min after reperfusion. In contrast, iNOS^‐/‐ mice experienced 35% mortality after reperfusion. The extensive myocardial injury was associated with marked apoptosis and infiltration of neutrophils whereas expression of HSP70 was less pronounced. Nitrotyrosine formation and plasma levels of nitrite and nitrate were undetectable. TNF‐α and IL‐6 were increased and IL‐10 was reduced in earlier stages of reperfusion. Activation of IKK and JNK and binding activity of NF‐κB and AP‐1 were significantly reduced. Thus, we conclude that iNOS plays a beneficial role in modulating the early defensive inflammatory response against reperfusion injury through regulation of signal transduction.—Zingarelli, B., Hake, P. W., Yang, Z., O’Connor, M., Denenberg, A., Wong, H. R. Absence of inducible nitric oxide synthase modulates early reperfusion‐induced NF‐κB and AP‐1 activation and enhances myocardial damage. FASEB J. 16, 327–342 (2002)