Several members of the Na⁺/H⁺exchanger gene family (NHE1, NHE2, NHE3, and NHE4) with unique functional properties have been cloned from rat epithelial tissues. The present study examined the molecular and pharmacological properties of Na⁺/H⁺exchange in rat parotid salivary gland cells. In acinar cells superfused with a physiological salt solution (145 mM Na⁺), Na⁺/H⁺exchanger activity was inhibited by low concentrations of the amiloride derivative ethylisopropyl amiloride (EIPA; IC₅₀ = 0.014 ± 0.005 μM), suggesting the expression of amiloride-sensitive isoforms NHE1 and/or NHE2. Semiquantitative RT-PCR confirmed that NHE1 transcripts are most abundant in this cell type. In contrast, the intermediate sensitivity of ductal cells to EIPA indicated that inhibitor-sensitive and -resistant Na⁺/H⁺exchanger isoforms are coexpressed. Ductal cells were about one order of magnitude more resistant to EIPA (IC₅₀ = 0.754 ± 0.104 μM) than cell lines expressing NHE1 or NHE2 (IC₅₀ = 0.076 ± 0.013 or 0.055 ± 0.015 μM, respectively). Conversely, ductal cells were nearly one order of magnitude more sensitive to EIPA than a cell line expressing the NHE3 isoform (IC₅₀= 6.25 ± 1.89 μM). Semiquantitative RT-PCR demonstrated that both NHE1 and NHE3 transcripts are expressed in ducts. NHE1 was immunolocalized to the basolateral membranes of acinar and ductal cells, whereas NHE3 was exclusively seen in the apical membrane of ductal cells. Immunoblotting, immunolocalization, and semiquantitative RT-PCR experiments failed to detect NHE2 expression in either cell type. Taken together, our results demonstrate that NHE1 is the dominant functional Na⁺/H⁺exchanger in the plasma membrane of rat parotid acinar cells, whereas NHE1 and NHE3 act in concert to regulate the intracellular pH of ductal cells.