Reductive carboxylation supports growth in tumor cells with defective mitochondria. Tumor cells with dysfunctional mitochondria rely on glutamine-dependent reductive carboxylation to generate citrate and acetyl-CoA for lipid synthesis and other metabolic processes. This pathway uses NADP⁺/NADPH-dependent isocitrate dehydrogenase (IDH) to convert glutamine-derived α-ketoglutarate into citrate, which is then used for lipid synthesis and other metabolic needs. This process is particularly important in cells with mutations in complex I or III of the electron transport chain (ETC), in patient-derived renal carcinoma cells with fumarate hydratase (FH) mutations, and in cells with normal mitochondria under acute ETC inhibition. The study used isogenic 143B human osteosarcoma cells with or without a loss-of-function mutation in ETC complex III to investigate metabolic changes. These cells showed a shift towards aerobic glycolysis and relied on glutamine for growth. Mass isotopomer analysis revealed that 143Bcytb cells, which lack functional ETC complex III, primarily used reductive carboxylation of glutamine-derived α-ketoglutarate to produce citrate, while 143Bwt cells used oxidative metabolism. This finding was confirmed in UOK262 cells, which lack FH activity and rely on glutamine-dependent reductive carboxylation for citrate and acetyl-CoA production. The study also showed that reductive carboxylation is a common response to impaired mitochondrial metabolism. When mitochondrial function is compromised, cells switch to reductive carboxylation to generate metabolic intermediates necessary for growth. This process is supported by IDH1 and IDH2, which are localized in the cytoplasm and mitochondria, respectively. Silencing these enzymes reduced cell growth, indicating their importance in tumor cell proliferation. The study further demonstrated that glutamine is the major carbon source for fatty acid synthesis in cells with impaired mitochondrial metabolism. Lipid analysis using 13C NMR showed that glutamine-derived carbon was the primary contributor to lipogenesis in 143Bcytb cells. This finding highlights the importance of glutamine in tumor cell growth, even when mitochondrial metabolism is impaired. Overall, the study reveals that reductive carboxylation is a critical metabolic pathway for tumor cells with defective mitochondria, enabling them to generate the necessary precursors for growth and survival. This pathway is particularly important in cells with mutations in the CAC or ETC, and it can be induced acutely during ETC inhibition. The findings have important implications for understanding tumor metabolism and developing targeted therapies for cancers with mitochondrial dysfunction.Reductive carboxylation supports growth in tumor cells with defective mitochondria. Tumor cells with dysfunctional mitochondria rely on glutamine-dependent reductive carboxylation to generate citrate and acetyl-CoA for lipid synthesis and other metabolic processes. This pathway uses NADP⁺/NADPH-dependent isocitrate dehydrogenase (IDH) to convert glutamine-derived α-ketoglutarate into citrate, which is then used for lipid synthesis and other metabolic needs. This process is particularly important in cells with mutations in complex I or III of the electron transport chain (ETC), in patient-derived renal carcinoma cells with fumarate hydratase (FH) mutations, and in cells with normal mitochondria under acute ETC inhibition. The study used isogenic 143B human osteosarcoma cells with or without a loss-of-function mutation in ETC complex III to investigate metabolic changes. These cells showed a shift towards aerobic glycolysis and relied on glutamine for growth. Mass isotopomer analysis revealed that 143Bcytb cells, which lack functional ETC complex III, primarily used reductive carboxylation of glutamine-derived α-ketoglutarate to produce citrate, while 143Bwt cells used oxidative metabolism. This finding was confirmed in UOK262 cells, which lack FH activity and rely on glutamine-dependent reductive carboxylation for citrate and acetyl-CoA production. The study also showed that reductive carboxylation is a common response to impaired mitochondrial metabolism. When mitochondrial function is compromised, cells switch to reductive carboxylation to generate metabolic intermediates necessary for growth. This process is supported by IDH1 and IDH2, which are localized in the cytoplasm and mitochondria, respectively. Silencing these enzymes reduced cell growth, indicating their importance in tumor cell proliferation. The study further demonstrated that glutamine is the major carbon source for fatty acid synthesis in cells with impaired mitochondrial metabolism. Lipid analysis using 13C NMR showed that glutamine-derived carbon was the primary contributor to lipogenesis in 143Bcytb cells. This finding highlights the importance of glutamine in tumor cell growth, even when mitochondrial metabolism is impaired. Overall, the study reveals that reductive carboxylation is a critical metabolic pathway for tumor cells with defective mitochondria, enabling them to generate the necessary precursors for growth and survival. This pathway is particularly important in cells with mutations in the CAC or ETC, and it can be induced acutely during ETC inhibition. The findings have important implications for understanding tumor metabolism and developing targeted therapies for cancers with mitochondrial dysfunction.