The sodium bicarbonate cotransporter (NBC1) mediates bicarbonate reabsorption in the renal proximal tubule. NBC1 activity is stimulated by 10% CO2, however, the mechanism is poorly understood. Here, we examined the mechanism of NBC1 regulation by 10% CO2 using an immortalized human proximal tubule cell line (HK2). In cells exposed to 10% CO2, the cotransporter activity (measured as deltapH/min) increased within minutes and this increase was maintained for 6 to 24 h. Early NBC1 stimulation was accompanied by increased NBC1 phosphorylation. Basolateral membrane NBC1 protein increased by 30 min and reached a maximum at 6 h. Increased NBC activity at 6 h was accounted for by increased NBC exocytosis to the basolateral membrane and not by decreased endocytosis. Latruncullin B (an actin cytoskeleton inhibitor) did not prevent CO2-induced stimulation, while nocodazole (a microtubule-disrupting agent) abrogated the stimulatory effect of 10% CO2. A significant increase in NBC1 mRNA expression level was observed at 6 h and maintained for 24 h. Total NBC1 protein increased at 12 to 24 h with 10% CO2 incubation and this effect was blocked by cycloheximide. In summary, the present study demonstrates that early activation of NBC1 activity by 10% CO2 was mediated by NBC1 phosphorylation. The stimulation of cotransporter activity observed at 6 h was due to exocytosis, while the late effect starting from 12 h was accounted for by increased protein synthesis.