Using the pH-sensitive fluorescent dye, 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester, we examined the effects of the hyperosmotic mannitol solution on basolateral Na⁺/H⁺ exchange (NHE) activity in the isolated nonperfused proximal tubule cells from the wild-type (WT) mice and those in which both the mdr1a and mdr1b genes were knocked out (KO). All the experiments were performed in CO₂/HCO₃^--free HEPES solutions. Osmolality of the peritubular solution was raised from 300 to 500 mOsm/kgH₂O by adding mannitol. The NHE activity was assessed by the Na⁺-dependent acid extrusion rates (J_H) after an acid load with NH₄Cl prepulse. In both groups of the cells under iso- and hyperosmotic conditions, we observed Na⁺-dependent pH_i increase that was significantly inhibited by pretreatment with ethylisopropylamiloride (the specific NHE inhibitor). Under isosmotic conditions, J_H at a wide pH_i range of 6.20 to 6.90 were not different between the two groups. The addition of the hyperosmotic mannitol to the cells of the WT mice had no effect on J_H at over the entire range of pH_i. In sharp contrast, in the cells of the KO mice, the hyperosmotic mannitol significantly increased J_H at pH_i values of 6.20 to 6.45, and shifted the J_H vs. pH_i by 0.15 pH units in the alkaline direction. The exposure of the hyperosmotic mannitol to the cells of the KO mice caused an increase in V_max without changing the K_m for peritubular Na⁺. The exposure of the cells of the WT mice to the hyperosmotic mannitol solution including cyclosporin A (the P-gp inhibitor) increased J_H (at pH_i of 6.30) with a similar magnitude to those of the exposure of the cells of the KO mice to the hyperosmotic mannitol alone. The addition of cyclosporin A to the cells of the KO mice had no effect on the hyperosmotic mannitol-induced increase in J_H. The exposure of the cells of the KO mice to either of the two protein kinase C (PKC) inhibitors (staurosporine or calphostin C) inhibited the hyperosmotic mannitol-induced increase in J_H. The stimulatory effect of the hyperosmotic mannitol on J_H was mimicked by the addition to the isosmotic control solution including phorbol 12-myristate 13-acetate (PMA, the PKC activator). The treatment of the cells from the WT mice with the hyperosmotic mannitol involving PMA increased J_H. We conclude: (a) Under isosmotic conditions, the basolateral membrane of the proximal tubule cells from both WT and KO mice possesses an NHE; (b) In the absence of P-gp activity, the hyperosmotic mannitol activates basolateral NHE via PKC, whereas in the presence of P-gp activity, it does not.