Using the pH-sensitive fluorescent dye BCECF, we compared the effects of hyperosmotic urea on basolateral Na⁺/H⁺ exchange (NHE) with those of hyperosmotic mannitol in isolated nonperfused proximal tubule S2 segments from mice lacking both the mdr1a and mdr1b genes (KO) and wild-type (WT) mice. All the experiments were performed in CO₂/HCO-free HEPES solutions. Osmolality of the peritubular solution was raised from 300 to 500 mosmol/kgH₂O by adding mannitol or urea. NHE activity was assessed by the Na⁺-dependent acid extrusion rate (J_H) after an acid load with NH₄Cl prepulse. In WT mice, hyperosmotic mannitol had no effect on J_H at over the entire range of intracellular pH (pH_i) studied (6.20-6.90), whereas in KO mice it increased J_H at a pH_i range of 6.20-6.45. In contrast, in both WT and KO mice, hyperosmotic urea increased J_H at a pH_i range of 6.20-6.90. In KO mice, J_H in a hyperosmotic urea solution were similar to those in a hyperosmotic mannitol solution at a pH_i range of 6.20-6.40 but were greater than in a hyperosmotic mannitol solution at a pH_i range of 6.45-6.90. In WT mice, hyperosmotic urea caused an increase in V_max without changing K_m for peritubular Na⁺. Staurosporine (the PKC inhibitor) inhibited hyperosmotic mannitol-induced NHE activation in KO mice, whereas it had no effect on hyperosmotic urea-induced NHE activation in WT or KO mice. Genistein (the tyrosine kinase inhibitor) inhibited hyperosmotic urea-induced NHE activation in WT and KO mice, whereas it caused no effect on hyperosmotic mannitol-induced NHE activation in KO mice. We conclude that hyperosmotic urea activates basolateral NHE via tyrosine kinase in tubules from both WT and KO mice, whereas hyperosmotic mannitol activates it via PKC only in tubules from KO mice.