Pancreatic cancer severity has been associated with high expression levels of extracellular carbonic anhydrases (CA), such as CAIX, and reduced expression of intracellular isoforms, such as CAII. The biochemical function of these enzymes is to catalyse the otherwise slow conversion between CO2 and HCO3- and H+ ions, but the role of this process in pancreatic cancer biology is unclear. CA activity was measured in five human pancreatic cancer cell lines: AsPC-1, PANC-1, BxPC3, MIA PaCa-2 and Colo357 (ranked in order of decreasing reliance on oxidative respiration, determined by effect of replacing glucose with galactose using CellTitre Blue viability assay). Intracellular CA activity (measured by cytoplasm-loaded SNARF pH-reporter dye upon rapid CO2/HCO3- addition/removal) was highest in AsPC-1 (9.98±0.54[SEM] above spontaneous, n>50) and decreased in the order PANC-1, Colo357, BxPC3 to MIA PaCa-2. In contrast, extracellular CA activity (measured by membrane-loaded exofacial WGA-fluorescein pH-reporter dye upon rapid NH3/NH4+ addition/removal) was highest in Colo357 (2.63±0.04 above spontaneous, n>50) and decreased in the order AsPC-1, BxPC3, MIA PaCa-2 to PANC-1. Chemical hypoxia (1 mM dimethyloxalylglycine) increased extracellular CA activity and CAIX expression in Colo357, AsPC-1 and BxPC-3 cells. Inhibition of CA (100 µM acetazolamide) in cell monolayers did not affect the growth after 72h (BCA protein assay; p>0.38[two tailed t-test], n=6), viability after 72h (CellTitre Blue; p>0.65, n=6) or colony formation after 21 days (clonogenic assay; p>0.55, n=3). This lack of effect may relate to the absence of large diffusion distances in thin monolayers, which are otherwise necessary to drive CO2/HCO3- out-of-equilibrium (the substrate for CA catalysis). To test whether extracellular CA activity may exert a physiologically role in diffusively-restricted tissue, Colo357 cells (which had the highest extracellular CA activity) were cultured as 3D tissue-growths (spheroids). Imaging intracellular pH confocally (SNARF) revealed a shallow surface-to-core gradient of intracellular pH (0.34±0.03 pH units, n=17) over a distance of 127±2.2μm (radius). Inhibition of CA activity with acetazolamide in size-matched spheroids (125±2.6μm) increased the degree of pH non-uniformity (0.50±0.09 pH units, n=11), and resulted in a significantly (p<0.01) more acidic spheroid-core. Thus, CA activity may only exert a biological effect in a diffusively-restricted environment of a spheroid, which resembles that in poorly-perfused solid tumours. Extracellular CA catalysis may be important for facilitating the venting of CO2 and lactic acid, particularly from the spheroid-core and thereby support higher metabolic and growth rates, essential for disease progression.