We have studied Cl^- transport mechanisms in freshwater rainbow trout gill mitochondrion-rich (MR) cells using intracellular pH (pH_i) imaging. Scanning electron microscopy demonstrated maintenance of cellular polarity in isolated MR cells. MR cell subtypes were identified by Na⁺ introduction to the bath, and Cl^- transport mechanisms were subsequently examined. Cl^- free exposure resulted in an alkalinization of pH_i in both MR cell subtypes, which was dependent on HCO₃^- in the bath and inhibited by 1 mM DIDS. Recovery of pH_i from an acidified state in Na⁺-free conditions was also DIDS sensitive. These results are the first functional evidence for Cl^-/HCO₃^- exchangers in fish gill MR cells. A direct switch from NaCl to Cl^- free conditions caused a pH_i acidification in a subset of MR cells, which was enhanced in the absence of HCO₃^-. The acidification was replaced by an alkalinization when Cl^- removal was performed in the presence of NPPB (500 µM) or EIPA (500 µM). Finally, we found that the Na⁺ induced alkalinization of pH_i found in a previous study is inhibited by EIPA. This inhibitor profile results suggest the presence of a Cl^- dependent Na⁺/H⁺ exchange mechanism.