The NBCn1 Na⁺/HCO₃^- cotransporter catalyze the electroneutral movement of 1 Na⁺:1 HCO₃^- into kidney cells. We characterized the intracellular pH (pH_i) regulation in human embryonic kidney cells (HEK) subjected to NH₄Cl prepulse acid loading, and we examined the NBCn1 expression and function in HEK cells subjected to 24-h elevated pCO₂ (10-15%). After acid loading, in the presence of HCO₃^-, ~50% of the pH_i recovery phase was blocked by the Na⁺/H⁺ exchanger (NHE) inhibitors EIPA (10-50 μM) and amiloride (1 mM), and was fully cancelled by 30 μM EIPA under nominally HCO₃^--free conditions. In addition, in the presence of HCO₃^-, pH_i recovery after acid loading was completely blocked when Na⁺ was omitted in the buffer. pH_i recovery after acidification in HEK cells was repeated in the presence of the NBC-inhibitor S0859, and the pH_i recovery was inhibit by S0859 in a dose-dependent manner (Ki= 30 μM, full inhibition at 60 μM), which confirmed NBC Na⁺/HCO₃^- cotransporter activation. NBCn1 expression increased threefold after 24-h exposure of cultured HEK cells to 10% CO₂ and sevenfold after exposure to 15% CO₂, examined by immunoblots. Finally, exposure of HEK cells to high CO₂ significantly increased the HCO₃^--dependent recovery of pH_i after acid loading. We conclude that HEK cells expressed the NBCn1 Na⁺/HCO₃^- cotransporter as the only HCO₃^--dependent mechanism responsible for cellular alkaline loading. NBCn1, which expresses in different kidney cell types, was upregulated by 24-h high CO₂ exposure of HEK cells, and this upregulation was accompanied by increased NBCn1-mediated HCO₃^- transport.