Resting or basal intracellular pH (pH_i) measured in cultured human syncytiotrophoblast cells was 7.26 ± 0.04 (without HCO3^-) or 7.24 ± 0.03 (with HCO3^-). Ion substitution and inhibitor experiments were performed to determine if common H⁺-transporting species were operating to maintain basal pH_i. Removal of extracellular sodium or chloride or addition of amiloride or H₂DIDS had no effect. Acidification using the K⁺/H⁺ exchanger, nigericin, reduced pH_i to 6.25 ± 0.15 (without HCO3^-) or 6.53 ± 0.10 (with HCO3^-). In the presence of extracellular sodium, recovery to basal pH_i was prompt and occurred at similar rates in the absence or presence of HCO3^-. Ion substitution and inhibition experiments were also used to identify the species mediating the return to basal pH_i following acidification. Recovery was inhibited by removal of sodium or addition of amiloride whereas removal of chloride or addition of H₂DIDS were ineffective. Addition of the Na⁺/H⁺ exchanger, monensin, to cells that had returned to basal pH_i elicited a further increase in pH_i, to 7.48 ± 0.07. Analysis of recovery data showed that there was a progressive decrease in pH/min as pH_i approached the basal level, despite the continued presence of a driving force for H⁺ extrusion. These data show that in cultured syncytial cells, in the absence of perturbation, basal pH_i is preserved despite the absence of active, mediated pH maintenance. They also demonstrate that an Na⁺/H⁺ antiporter acts to defend the cells against acidification and that it is the sole transporter necessary for recovery from an intracellular acid load.