Heterogenous metabolic microenvironments in tumours affect local cell growth, survival, and overall therapeutic efficacy. Hypoxia-inducible-factor-1 alpha (HIF1-alpha) is a transcription factor that responds to low-oxygen environments by upregulating genes for cell survival and metabolism. To date, the metabolic effects of HIF-1 alpha in three-dimensional tissue have not been investigated. Preliminary experiments have shown that effects of HIF-1 alpha are dependent on glucose availability. Based on this observation, we hypothesized that HIF-1 alpha would not affect cell survival and metabolism in the center of spheroids, where the concentrations of oxygen and glucose are low, similar to hypoxic regions found in tumors. To test this hypothesis we used fluorescence microscopy and the tumor cylindroid model to quantify cellular viability in three-dimensional tissue. Isotope labeling and metabolic flux analysis were also used to quantify the intracellular metabolism of wild-type and HIF-1 alpha-null spheroids. As hypothesized, cell survival and intracellular metabolism were not different between wild-type and HIF-1 alpha-null tissues. In addition, small spheroids, which contain less quiescent cells and are less nutritionally limited, were found to have increased carbon flux through the biosynthetic pentose and pyruvate carboxylase pathways. These results show nutrient gradients affect cell growth and metabolism in spheriods and suggest that metabolic microenvironment should be taken into account when developing HIF-1 alpha-based therapies.