T o understand the role of Na⁺/H⁺ exchanger 1 (NHE1) in intracellular pH (pH_i) regulation and neuronal function, we took advantage of natural knockout mice lacking NHE1, the most ubiquitously and densely expressed NHE isoform in the central nervous system (CNS). CA1 neurons from both wild-type (WT) and NHE1 mutant mice were studied by continuous monitoring of pH_i, using the fluorescent indicator carboxy-seminaphthorhodafluor-1 (SNARF-1) and confocal microscopy. In the nominal absence of CO₂/HCO₃^–, steady-state pH_i was higher in WT neurons than in mutant neurons. Using the NH₄Cl prepulse technique, we also show that H⁺ flux in WT neurons was much greater than in mutant neurons. The recovery from acid load was blocked in WT neurons, but not in mutant neurons, by removal of Na⁺ from the extracellular solution or by using 100 μM 3-(methylsulfonyl-4-piperidino-benzoyl)-guanidine methanesulfonate (HOE 694) in HEPES buffer. Surprisingly, in the presence of CO₂/HCO₃^–, the difference in H⁺ flux between WT and mutant mice was even more exaggerated, with a difference of more than 250 μM/s between them at pH 6.6. H⁺ flux in CO₂/HCO₃^– was responsive to diisothiocyanato-stilbene-2,2′-disulfonate (DIDS) in the WT but not in the mutant. We conclude that (a) the absence of NHE1 in the mutant neurons tended to cause lower steady-state pH_i and, perhaps more importantly, markedly reduced the rate of recovery from an acid load; and (b) this difference in the rate of recovery between mutant and WT neurons was surprisingly larger in the presence, rather than in the absence, of HCO₃^–, indicating that the presence of NHE1 is essential for the regulation and/or functional expression of both HCO₃^–-dependent and -independent transporters in neurons.