Multicellular spheroids, an appropriate in vitro system for simulating 3-D tumor micro-milieu can be used for evaluating and predicting tumor response to therapeutic agents including metabolic inhibitors. However, detailed understanding of the nature, distribution and sensitivity/responses of cellular sub-populations to potential therapeutic agents/strategies is required for using this unique model with optimal precision. Spheroid characteristics may also vary considerably with the origin and type of cell line used, and thorough characterization of viable and dissociated glioma cell spheroids is not yet completely known. In order to evaluate in vivo responses of gliomas to various therapeutic strategies, especially the metabolic inhibitors capable of penetrating the blood brain barrier, we have characterized continuously growing spheroids of a human glioma cell line (BMG-1) with respect to organization, growth, viability, cell survival, cell death, metabolic and mitochondrial status, oxidative stress and radiation response using microscopy, flow cytometry and enzymatic assays. Spheroids were fed daily with fresh medium in order to maintain nutrient supply to outer cellular layers while hypoxia/necrosis developed in the innermost cells of enlarging spheroids. Volume of spheroids, fed daily with fresh medium, increased exponentially during 7-28 days of growth through three population doublings. Proportion of G1-phase cells was higher (approximately 60%) than exponentially growing monolayer cells (approximately 48%). A significant fraction of S-phase cells turned metabolically inactive (disengaged in DNA synthesis) with increasing age of the spheroids, unlike in quiescent monolayer cultures, where the fraction of S-phase cells was less than 5%. With increasing spheroid size, increasing sub-populations of cells became non-viable and entered apoptosis or necrosis revealed by Annexin-V-FITC/PI staining. PI positive (necrotic) cells were not confined to the centre of the spheroid, but distributed at certain discrete foci. Average glucose consumption and lactate production were 2-3 folds higher in viable spheroid cells compared to monolayer cells, implying a compensatory increase in glycolysis possibly due to hypoxic environment. HIF-1alpha was expressed only in spheroids and increased in an age-dependent manner, whereas c-Myc (known to induce apoptosis in glucose-deprived cells) levels were three times higher than monolayer cells. Mitochondrial mass and activity decreased significantly during first 14 days of growth but increased with age, and were not associated with increase in ROS levels. Bcl-2 and Bax levels were higher (approximately 2 folds) than monolayers, while the ratio (Bcl/Bax) remained unaltered. Radiation-induced oxidative stress was considerably less in spheroids as compared to monolayers, and corresponded well with increase in radioresistance demonstrated by the clonogenic assay, similar to hypoxia induced radioresistance observed in tumors. Development of S-negative cells and reduced endogenous and radiation-induced ROS coupled with higher levels of anti (Bcl2) as well as pro (Bax) apoptotic regulators observed in spheroids suggest the intricate/complex nature of endogenous as well as induced stress resistance that could exist in tumors, which contribute to the treatment resistance.