H+ ions are highly reactive and potent modulators of cellular physiology. Out of convention, their concentration is typically expressed using a logarithmic pH scale. Cells must regulate their intracellular pH (pHi) in order to produce a milieu that is conducive for biochemical reactions, many of which work optimally only within a narrow pH range. However, cellular respiration can disturb pHi balance by loading the cytoplasm with CO2 and lactic acid. If the cellular removal of these products is inadequate, their intracellular spontaneous ionisation can reduce steady-state pHi significantly. Membrane-bound transporters, such as Na+/H+ exchange, can remove this excess of H+ ions. The process of acid-removal is then completed by the diffusion of extruded H+ ions away from the respiring cell. This is important for maintaining a constant extracellular pH (pHe). An alternative to trans-membrane H+-flux is the transport of HCO3- ions into the cell by means of proteins such as Na+-HCO3- cotransport. Imported HCO3- ions react with excess intracellular H+ ions, producing the membrane-permeant product, CO2. Diffusion of CO2 across the membrane and away from the cell completes the process of acid-extrusion. Regulation of pHi is therefore a process involving buffering reactions, membrane transport and diffusion. Tumours represent an unusual challenge to pHi regulation because of their high metabolic rate (hence elevated acid-production), typically inadequate blood perfusion (hence weaker cell-capillary diffusive coupling) and a high proliferative rate that requires an alkaline pHi. Cancer tissue has been shown to express high levels of the membrane-tethered, extracellular-facing enzyme carbonic anhydrase IX (CAIX; Pastorek et al., 1994). It has been speculated that this enzyme is important for pH regulation because it catalyses the reversible reaction CO2 + H2O = H+ + HCO3-, which involves H+ ions and the components of CO2/HCO3- buffer. We have studied the role of CAIX in three-dimensional, multi-cellular growths (radius 100-300 μm; made of 10,000-200,000 cells) cultured from cancer-derived cell lines, and imaged confocally for pHi with carboxy-SNARF-1 or pHe with membrane-impermeant fluorescein-derivatives. Using spheroids grown from renal bladder RT112 and colonic HCT116 cells, we have shown that CAIX activity facilitates CO2 venting from cells (Swietach et al, 2008; 2009). Spheroids made of cells over-expressing CAIX had smaller radial pHi gradients, with notably less acidic pHi at the core, compared to spheroids treated with CA inhibitors or spheroids made of sham-transfected cells. CAIX can facilitate CO2 efflux by hydrating extracellular CO2 and thereby maintaining a steep CO2 efflux gradient. Using HCT116 spheroids, we have confirmed that CAIX activity lowers pHe by releasing H+ ions extracellularly (Swietach et al, 2009). This process may contribute towards the low pHe typically measured in tumours. Moreover, low pHe has been proposed to underlie the selection pressure favouring the more pHe-tolerant cancer cells over normal cells. The other product of CAIX-catalysed CO2 hydration, HCO3-, can be taken-up by cells to regulate pHi. We have shown that HCT116, RT112, colon HT29, breast MDA-mb-468, cervical HeLa and pancreatic MiaPaca2 cells produce a significant HCO3–flux that regulates pHi in parallel with H+-flux. The magnitude of HCO3–flux was less variable than that of H+-flux. HCO3- transport may therefore represent a constitutive element of pHi regulation, important for cancer pHi physiology (Hulikova et al, 2011). In some cell lines, notably HCT116 and MDA-mb-468, H+-flux was greater than HCO3–flux. However, spheroids grown from these cells were unable to regulate pHi in the absence of extracellular CO2/HCO3- buffer. Mobile buffers such as CO2/HCO3- facilitate H+ ion diffusion in the unstirred extracellular space of tumours. Without this H+ ion chaperoning, sustained extrusion of H+ ions from cells would reduce pHe to levels that may inhibit membrane-bound pHi regulators. To maximise the H+ ion shuttling capacity of CO2/HCO3- buffer, the protonation and deprotonation kinetics must be catalysed by CA activity. We have shown that H+-flux in MDA-mb-468 spheroids was reduced significantly in the presence of CA inhibitors. In summary, CAIX serves an important role in tumour pHi regulation by (i) facilitating trans-membrane CO2 efflux, (ii) supplying substrate for HCO3–dependent pHi regulating proteins, and (iii) facilitating extracellular H+ ion mobility. Inhibition of CAIX activity may disrupt the powerful pHi regulatory apparatus in cancer and provide a novel target for therapeutic interventions.