Active metabolic pathways in three-dimensional cancer-cell cultures are potential chemotherapeutic targets that would he effective throughout tumors. Chaotic vasculature creates cellular regions in tumors with distinct metabolic behavior that are only present in aggregate cell masses. To quantify cancer cell metabolism, transformed mouse fibroblasts were grown as spheroids and fed isotopically labeled culture medium. Metabolite uptake and production rates were measured as functions of time. Gas chromatography-mass spectrometry was used to quantify the extent of labeling on amino acids present in cytoplasmic,extracts. The labeling pattern identified several active and inactive metabolic pathways: Glutaminolysis was found to be active, and malic enzyme and gluconeo genesis were inactive. Transformed cells in spheroids were also found to actively synthesize serine,. cysteine, alanine, aspartate, glutamate, and proline; and not synthesize glutamine. The activities of these pathways suggest that cancer cells consume glutamine for biosynthesis and not to provide cellular energy. Determining active metabolic pathways indicates how cells direct carbon flow and may lead to the discovery of novel molecular targets for anticancer therapy. (C) 2009 American Institute of Chemical Engineers Biotechnol. Prog., 26: 789-796, 2010