Acid-extrusion by membrane-bound transporter-proteins is an essential pH-regulatory mechanism that protects cells from excessive acidification. This is particularly important in cancer cells, which produce large quantities of acid by virtue of their high metabolic rate. Signaling cascades can regulate acid-extruding transporters through gene expression, post-translational modifications or changes in the chemical milieu, which then affect transport rate. Hypoxia is a major trigger of adaptive responses in cancer and plays an important role in disease progression [1]. However, the effect of hypoxic signaling on acid-extrusion remains unclear. Acid-extrusion was measured in eight cancer-derived cell-lines (HCT116, RT112, MDA-MB-468, MCF10A, HT29, HT1080, MiaPaca2, HeLa) using the pH-sensitive dye, cSNARF-1 [2]. Hypoxic signaling was triggered by pre-incubation in low (1-2%) O2 or with the 2-oxoglutarate-dependent dioxygenase inhibitor dimethyl-oxalylglycine (DMOG). By means of selective pharmacological inhibition (30µM dimethylamiloride to block NHE1 activity) or transport-substrate removal (Na+, HCO3−), acid-extrusion flux was dissected into components due mainly to Na+/H+ exchange (NHE) and HCO3− transport. In HCT116 cells, hypoxia-triggered signaling reduced NHE-flux reversibly (by 51±8% with a time-constant of 1.8 hrs for 1% O2; by 64±4% with a time-constant of 1.1 hrs for 1mM DMOG). This was associated with a change in NHE1 protein phosphorylation (consensus sequence substrates for protein kinases A and C) but not NHE1 expression or internalization (studied with monoclonal NHE1 antibody) or ATP levels (luciferase assay). NHE-flux remained reduced (by ~53%) following a 48-hour activation of hypoxic pathways (2% O2 or 1mM DMOG), but the effect was associated with reduced expression of the mature form of NHE1 and was slowly reversible. These hypoxic responses of acid-extrusion in HCT116 cells were also observed in RT112 and MCF10A cells. Acid-extrusion by NHE was not hypoxia-sensitive in MDA-MB-468 cells, whereas in the remaining cell-lines, normoxic NHE activity was too low to resolve for hypoxic-response. HCO3− transport, in contrast, was not significantly affected by low O2 or DMOG pre-incubation in any of the cell-lines studied. Thus, HCO3− transport emerges as a constitutive and stable element of pH regulation, whereas NHE flux can vary widely in magnitude between different cell-lines and their hypoxic status. Heterogeneity of O2 tension in tumors can produce complex and important effects on the ability of cells to regulate pH. Different degrees of hypoxia-sensitivity may underlie adaptive responses in cancer, and be of significance to therapeutic approaches targeting pH-regulating proteins.