To assess the role of Ca²⁺ in regulation of the Na⁺/H⁺ exchanger (NHE1), we used CCL-39 fibroblasts overexpressing the Na⁺/Ca²⁺ exchanger (NCX1). Expression of NCX1 markedly inhibited the transient cytoplasmic Ca²⁺ rise and long-lasting cytoplasmic alkalinization (60-80% inhibition) induced by -thrombin. In contrast, coexpression of NCX1 did not inhibit this alkalinization in cells expressing the NHE1 mutant with the calmodulin (CaM)-binding domain deleted (amino acids 637-656), suggesting that the effect of NCX1 transfection involves Ca²⁺-CaM binding. Expression of NCX1 only slightly inhibited platelet-derived growth factor BB-induced alkalinization and did not affect hyperosmolarity- or phorbol 12-myristate 13-acetate-induced alkalinization. Downregulation of protein kinase C (PKC) inhibited thrombin-induced alkalinization partially in control cells and abolished it completely in NCX1-transfected cells, suggesting that the thrombin effect is mediated exclusively via Ca²⁺ and PKC. On the other hand, deletion mutant study revealed that PKC-dependent regulation occurs through a small cytoplasmic segment (amino aids 566-595). These data suggest that a mechanism involving direct Ca²⁺-CaM binding lasts for a relatively long period after agonist stimulation, despite apparent short-lived Ca²⁺ mobilization, and further support our previous conclusion that Ca²⁺- and PKC-dependent mechanisms are mediated through distinct segments of the NHE1 cytoplasmic domain.