The effect of CO₂/HCO₃⁻ on the endocochlear potential (EP) was examined by using both ion-selective and conventional microelectrodes and the endolymphatic or perilymphatic perfusion technique. The main findings were as follows: (i) A decrease in the EP from ∼+75 to ∼+35 mV was produced by perilymphatic perfusion with CO₂/HCO₃⁻-free solution, which decrease was accompanied by an increase in the endolymphatic pH (ΔpH_e, ∼0.4). (ii) Perilymphatic perfusion with a solution containing 20 mM NH₄Cl produced a decrease in the EP (ΔEP, ∼20 mV) with an increase in the pH_e (ΔpH_e, ∼0.2), whereas switching the perfusion solution from the NH₄Cl solution to a 5% CO₂/25 mM HCO₃⁻ solution produced a gradual increase in the EP to the control level with the concomitant recovery of the pH_e. (iii) The perfusion with a solution of high or low HCO₃⁻ with a constant CO₂ level within 10 min produced no significant changes in the EP. (iv) Perfusion of the perilymph with 10 \\xb5 g/ml nifedipine suppressed the transient asphyxia-induced decrease in EP slightly, but not significantly. (v) By contrast, the administration of 1 μg/ml nifedipine via the endolymph inhibited significantly the reduction in the EP induced by transient asphyxia or perilymphatic perfusion with CO₂/HCO₃⁻-free or 20 mM NH₄Cl solution. These findings suggest that the effect of CO₂ removal from perilymphatic perfusion solution on the EP may be mediated by an increase in cytosolic Ca²⁺ concentration induced by an elevation of cytosolic pH in endolymphatic surface cells.