The study investigates the metabolic pathways used by tumor cells with defective mitochondria to generate precursors for macromolecular synthesis. Specifically, it focuses on tumor cells lacking function in the citric acid cycle (CAC) or electron transport chain (ETC), which impair normal oxidative mitochondrial function. The researchers found that these tumor cells use glutamine-dependent reductive carboxylation as the primary pathway for citrate formation, rather than oxidative metabolism. This pathway involves mitochondrial and cytosolic isoforms of NADP+/NADPH-dependent isocitrate dehydrogenase (IDH) and subsequent metabolism of glutamine-derived citrate to produce acetyl-CoA for lipid synthesis and 4-carbon intermediates for other CAC metabolites. This reductive, glutamine-dependent pathway is dominant in rapidly growing malignant cells with mutations in complex I or complex III of the ETC, in patient-derived renal carcinoma cells with fumarate hydratase (FH) mutations, and in cells with normal mitochondria subjected to acute pharmacological ETC inhibition. The findings reveal a novel metabolic strategy that supports tumor cell growth and explains how cells generate CAC intermediates despite impaired mitochondrial function.The study investigates the metabolic pathways used by tumor cells with defective mitochondria to generate precursors for macromolecular synthesis. Specifically, it focuses on tumor cells lacking function in the citric acid cycle (CAC) or electron transport chain (ETC), which impair normal oxidative mitochondrial function. The researchers found that these tumor cells use glutamine-dependent reductive carboxylation as the primary pathway for citrate formation, rather than oxidative metabolism. This pathway involves mitochondrial and cytosolic isoforms of NADP+/NADPH-dependent isocitrate dehydrogenase (IDH) and subsequent metabolism of glutamine-derived citrate to produce acetyl-CoA for lipid synthesis and 4-carbon intermediates for other CAC metabolites. This reductive, glutamine-dependent pathway is dominant in rapidly growing malignant cells with mutations in complex I or complex III of the ETC, in patient-derived renal carcinoma cells with fumarate hydratase (FH) mutations, and in cells with normal mitochondria subjected to acute pharmacological ETC inhibition. The findings reveal a novel metabolic strategy that supports tumor cell growth and explains how cells generate CAC intermediates despite impaired mitochondrial function.