Recent investigation has focused on identifying signaling pathways that inhibit cardiac hypertrophy, a major risk factor for cardiovascular morbidity and mortality. In this context, nitric oxide (NO), signaling via cGMP and cGMP-dependent protein kinase type I (PKG I), has been recognized as a negative regulator of cardiac myocyte (CM) hypertrophy. However, the underlying mechanisms are poorly understood. Here, we show that PKG I inhibits CM hypertrophy by targeting the calcineurin-NFAT signaling pathway. Calcineurin, a Ca 2؉ -dependent phosphatase, promotes hypertrophy in part by activating NFAT transcription factors which induce expression of hypertrophic genes, including brain natriuretic peptide (BNP). C ardiac hypertrophy has been viewed as a compensatory mechanism of the heart that helps to maintain cardiac output during pathological states with sustained increases in hemodynamic load. However, hypertrophy often heralds decompensation, transition to heart failure, and sudden death (1). To understand the molecular mechanisms of this ultimately maladaptive response, much investigation has focused on the signaling pathways controlling hypertrophy at the level of the single cardiac myocyte (CM). It has emerged that the hypertrophic response is orchestrated by growth factors and cytokines acting through several interdependent signaling cascades, including, for example, specific G protein isoforms, low-molecular-weight GTPases, mitogen-activated protein kinases, and protein kinase C (2, 3). In addition, recent studies have recognized the importance of Ca 2ϩ -sensitive signaling pathways in CM hypertrophy (4). One prominent Ca 2ϩ -dependent pathway involves the Ser͞ Thr protein phosphatase calcineurin. Activation of calcineurin by Ca 2ϩ results in the dephosphorylation and nuclear translocation of cytoplasmic latent nuclear factor of activated T cells (NFAT) transcription factors (5). Many lines of evidence, including the antihypertrophic effects of endogenous calcineurin inhibitory peptides, have contributed to the conclusion that the calcineurin-NFAT pathway plays an essential role in the hypertrophic response to growth factor stimulation (6-9). Importantly, calcineurin activates and interacts with other hypertrophic signaling cascades (10-13), supporting the concept that CM hypertrophy is controlled by crosstalking signaling networks.Adding yet another level of complexity, recent studies have identified signaling pathways opposing CM hypertrophy (14-16). In this context, the free radical gas nitric oxide (NO) has been recognized as a negative regulator of the hypertrophic response (17)(18)(19)(20). Antihypertrophic effects of NO are mediated via the second messenger cGMP and its activation of cGMP-dependent protein kinase type I (PKG I) (19, 21), although the mechanisms involved have not been elucidated. Specifically, it is not known whether PKG I exerts antihypertrophic effects via an autonomous inhibitory pathway and͞or via suppression of certain hypertrophic signaling cascades. Because PKG I regulates [Ca 2...