The cardiac Na(+)/Ca(2+) exchanger (NCX1) is the predominant mechanism for the extrusion of Ca(2+) from beating cardiomyocytes. The role of protein phosphorylation in the regulation of NCX1 function in normal and diseased hearts remains unclear. In our search for proteins that interact with NCX1 using a yeast two-hybrid screen, we found that the C terminus of calcineurin Abeta, containing the autoinhibitory domain, binds to the beta1 repeat of the central cytoplasmic loop of NCX1 that presumably constitutes part of the allosteric Ca(2+) regulatory site. The association of NCX1 with calcineurin was significantly increased in the BIO14.6 cardiomyopathic hamster heart compared with that in the normal control. In hypertrophic neonatal rat cardiomyocytes subjected to chronic phenylephrine treatment, we observed a marked depression of NCX activity measured as the rate of Na(+)(i)-dependent (45)Ca(2+) uptake or the rate of Na(+)(o)-dependent (45)Ca(2+) efflux. Depressed NCX activity was partially and independently reversed by the acute inhibition of calcineurin and protein kinase C activities with little effect on myocyte hypertrophic phenotypes. Studies of NCX1 deletion mutants expressed in CCL39 cells were consistent with the view that the beta1 repeat is required for the action of endogenous calcineurin and that the large cytoplasmic loop may be required to maintain the interaction of the enzyme with its substrate. Our data suggest that NCX1 is a novel regulatory target for calcineurin and that depressed NCX activity might contribute to the etiology of in vivo cardiac hypertrophy and dysfunction occurring under conditions in which both calcineurin and protein kinase C are chronically activated.