Intracellular pH (pHi), a major modulator of cell function, is regulated by acid/base transport across membranes. Excess intracellular H+-ions (e.g. produced by respiration) are extruded by transporters such as Na+/H+ exchange, or neutralized by HCO3- taken-up by carriers such as Na+-HCO3- cotransport. Using fluorescence pHi-imaging, we show that cancer-derived cell-lines (colorectal HCT116 and HT29, breast MDA-MB-468, pancreatic MiaPaca2, cervical HeLa) extrude acid by H+-efflux and HCO3−-influx, largely sensitive to 30 μM dimethylamiloride (DMA) and 100 μM 4,4′-diisothiocyanatostilbene-2,2′-disulfonate (DIDS), respectively. The magnitude of HCO3–influx was comparable among the cell-lines, and may represent a constitutive element of tumor pHi regulation (3.0±0.4[SEM] to 4.3±0.4 mM/min at pHi=6.7). In contrast, H+-efflux varied considerably (MDA-MB-468>HCT116>HT29>MiaPaca2> HeLa; from 7.0±0.4[SEM] to 0.7±0.2[SEM] mM/min at pHi=6.7). When HCO3–flux was pharmacologically inhibited with DIDS, acid-extrusion in multi-cellular HT29 and HCT116 spheroids (radii 130-200 μm; ~10,000-50,000 cells) was highly non-uniform and produced low pHi at the core (see Figure). With depth, acid-extrusion became relative more DIDS-sensitive because the low extracellular pH at the spheroid core inhibits H+-flux more than HCO3–flux. HCO3–flux inhibition also decelerated HCT116 spheroid growth. In the absence of CO2/HCO3-, acid-extrusion by H+-flux in HCT116 and MDA-MB-468 spheroids became highly non-uniform and inadequate at the core (see Figure). This is because H+-transporters require extracellular mobile pH-buffers, like CO2/HCO3-, to overcome low H+-ion mobility and chaperone H+-ions away from cells. The effect of 5% CO2/22 mM HCO3- was mimicked by 30 mM Hepes (an artificial buffer system) but reduced when carbonic anhydrase activity (which normally keeps CO2/HCO3- at equilibrium) was inhibited with acetazolamide (100 μM; see Figure). CO2/HCO3- exerts a dual effect: as substrate for membrane-bound HCO3–transporters, and as a mobile buffer for facilitating extracellular diffusion of H+-ions extruded from cells. These processes can be augmented by carbonic anhydrase activity. We conclude that CO2/HCO3- is important for maintaining uniformly alkaline pHi in small, non-vascularized tumor-growths and may be important for cancer disease progression.