Using the pH-sensitive fluorescent dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester, we examined the effects of hyperosmotic mannitol on basolateral Na⁺/H⁺ exchange (NHE) activity in isolated nonperfused proximal tubule S2 segments from mice lacking both the mdr1a and mdr1b genes (KO) and wild-type mice (WT). All experiments were performed in CO₂/HCO-free HEPES solutions. Osmolality of the peritubular solution was raised from 300 to 500 mosmol/kgH₂O by the addition of mannitol. NHE activity was assessed by Na⁺-dependent acid extrusion rates (J_H) after an acid load with NH₄Cl prepulse. Under isosmotic conditions, J_H values at a wide intracellular pH (pH_i) range of 6.20-6.90 were not different between the two groups. In WT mice, hyperosmotic mannitol had no effect on J_H at the wide pH_i range. In contrast, in KO mice, hyperosmotic mannitol increased J_H at a pH_i range of 6.20-6.45 and shifted the J_H-pH_i relationship by 0.15 pH units in the alkaline direction. In KO mice, hyperosmotic mannitol caused an increase in maximal velocity without changing the Michaelis-Menten constant for peritubular Na⁺. Exposure of cells from WT mice to the hyperosmotic mannitol solution including the P-gp inhibitor cyclosporin A increased J_H (at pH_i 6.30) to an extent similar to that in cells from KO mice exposed to hyperosmotic mannitol alone. In KO mice, staurosporine and calphostin C inhibited the hyperosmotic mannitol-induced increase in J_H. The stimulatory effect of hyperosmotic mannitol on J_H was mimicked by addition to the isosmotic control solution, including phorbol 12-myristate 13-acetate (PMA; the PKC activator). In WT mice, hyperosmotic mannitol with PMA increased J_H. We conclude that, in the absence of P-gp activity, hyperosmotic mannitol activates basolateral NHE via protein kinase C, whereas in the presence of P-gp activity, it does not.