Transgenic overexpression of calcineurin (CN/Tg) in mouse cardiac myocytes results in hypertrophy followed by dilation, dysfunction and sudden death. Nitric oxide produced via inducible nitric oxide synthase (iNOS) has been implicated in cardiac injury. Since calcineurin regulates iNOS expression and since phenotypes of mice overexpressing iNOS are similar to CN/Tg, we hypothesized that iNOS is pathogenically involved in cardiac phenotypes of CN/Tg mice. CN/Tg mice had increased serum and cardiac iNOS levels. When CN/Tg-iNOS -/- and CN/Tg mice were compared some phenotypes were similar: extent of hypertrophy and fibrosis. However, CN/Tg-iNOS -/- mice had: improved systolic performance (p<0.001), less heart block (p<0.0001); larger INa density, lower serum TNF levels (p<0.03); and less apoptosis (p<0.01) resulting in improved survival (p<0.0003). To define tissue origins of iNOS production, chimeric lines were generated. Bone marrow (BM) from wild-type or iNOS -/- mice was transplanted into CN/Tg mice. iNOS deficiency restricted to BM-derived cells was not protective. Calcineurin activates local production of NO by iNOS in cardiac myocytes which significantly contributes to sudden death, heart block, LV dilation and impaired systolic performance in this murine model of cardiac hypertrophy induced by overexpression of calcineurin.