Solid tumors contend with, and adapt to, a hostile micro-environment that includes limited availability of nutrient fuels and oxygen. The presence of hypoxia (O-2 <5%) stabilizes the transcription factor Hif1 and results in numerous cellular adaptations including increased flux of glucose through glycolysis. Increasingly, more sophisticated analysis of tumor oxygenation has revealed large gradients of oxygen tension and significant regions under severe hypoxia (O-2 approximate to 0.1%). The present investigation has demonstrated a significant increase in the glycolytic flux rate when tumor spheroids were exposed to 0.1% O-2. The severe hypoxia was associated with uniform pimonidazole adduct formation and elevated levels of Hif1 and c-Myc. This resulted in elevated expression of GLUT and MCT transporters, in addition to increased activity of PFK1 in comparison to that observed in normoxia. However, the protein expression and enzymatic capacity of HK2, G6PDH, PK, and LDH were all reduced by severe hypoxia. Clearly, the effects of exposure to severe hypoxia lead to a significantly abridged Hif1 response, yet one still able to elevate glycolytic flux and prevent loss of intermediates to anabolism. J. Cell. Biochem. 117: 1890-1901, 2016. (c) 2016 Wiley Periodicals, Inc.