The calcineurin A (CaNA) subunit was identified as a novel binding partner of plasma membrane Na(+)/H(+) exchanger 1 (NHE1). CaN is a Ca(2+)-dependent phosphatase involved in many cellular functions, including cardiac hypertrophy. Direct binding of CaN to the (715)PVITID(720) sequence of NHE1, which resembles the consensus CaN-binding motif (PXIXIT), was observed. Overexpression of NHE1 promoted serum-induced CaN/nuclear factor of activated T cells (NFAT) signaling in fibroblasts, as indicated by enhancement of NFAT promoter activity and nuclear translocation, which was attenuated by NHE1 inhibitor. In neonatal rat cardiomyocytes, NHE1 stimulated hypertrophic gene expression and the NFAT pathway, which were inhibited by a CaN inhibitor, FK506. Importantly, CaN activity was strongly enhanced with increasing pH, so NHE1 may promote CaN/NFAT signaling via increased intracellular pH. Indeed, Na(+)/H(+) exchange activity was required for NHE1-dependent NFAT signaling. Moreover, interaction of CaN with NHE1 and clustering of NHE1 to lipid rafts were also required for this response. Based on these results, we propose that NHE1 activity may generate a localized membrane microdomain with higher pH, thereby sensitizing CaN to activation and promoting NFAT signaling. In cardiomyocytes, such signaling can be a pathway of NHE1-dependent hypertrophy.