As a major modulator of cardiac function, intracellular pH (pHi) is tightly controlled by sarcolemmal acid-base transporters to within narrow limits (7.1-7.3). Na+/H+ exchanger (NHE1) and Na+/HCO3 - cotransporter (NBC) are the main acid-extruding membrane proteins; the latter is further subdivided into electrogenic (NBCe1/NBCe2) and electroneutral (NBCn1) isoforms. In the underperfused heart, acid disturbances are often accompanied by hypoxia, but their interplay on cardiac NBC activity is unknown. Here, we studied the effect of acute (1 mM dithionite and 100% N2, 10 min) and long-term hypoxia (1% O2, 48 h) on sarcolemmal NBC activity using fluorimetric assays in mouse atrial-derived HL-1 cells and primary rat cardiomyocytes. NBCe1 and NBCn1 transcripts were detected in HL-1 cells. Ensemble NBC activity, defined as the HCO3 --dependent acid-extrusion flux, was promptly inhibited under acute anoxia. In contrast, pHi-sensitivity of NBC flux was increased after long-term hypoxia, likely an adaptive response. This increase was not due to buffering capacity changes but was mimicked by dimethyloxalylglycine (1 mM, DMOG), which stabilizes hypoxia inducible factor under normoxic conditions. Hypoxia affected neither NBCn1 nor NBCe1 protein levels, indicating a modulatory effect on transporter activity. The contribution of electrogenic (NBCe1) and electroneutral (NBCn1) isoforms, dissected from fluxes generated under hyperkalemia, showed that long-term hypoxia selectively raised NBCn1 activity. This effect was blocked by U0126, an inhibitor of extracellular signal-regulated kinase 1/2, implicating phosphorylation. Our results show that acute anoxia and prolonged hypoxia regulate NBC-dependent flux by distinct mechanisms ostensibly to retain pH control under the combination of acidosis and hypoxia.