One-carbon (1C) metabolism, supported by the folate cofactor, is essential for various physiological processes, including biosynthesis of purines and thymidine, amino acid homeostasis, epigenetic maintenance, and redox defense. This metabolism is compartmentalized within eukaryotic cells and across organs. The review highlights recent discoveries in mammalian 1C metabolism, emphasizing the importance of mitochondrial 1C reactions in producing 1C units for the cytosol and generating additional products like glycine and NADPH. It discusses the differential use of folate pathways in cancer, stem cells, development, and adult physiology, offering new therapeutic opportunities. Folate metabolism involves a complex set of molecules with a common core structure, including a pteridine ring, a para-aminobenzoic acid (PABA) linker, and a polyglutamate tail. The biologically active form of folate is tetrahydrofolate (THF), which carries 1C units for biosynthetic processes. 5,10-methylene-THF is used by thymidylate synthase to convert dUMP to dTMP, and by serine hydroxymethyltransferase (SHMT) to convert glycine to serine. 5-methyl-THF is involved in homocysteine remethylation to form methionine, which is crucial for S-adenosylmethionine (SAM) synthesis, a key methyl donor in epigenetics and other biosynthetic processes. 10-formyl-THF is essential for purine synthesis and can be converted to formate or fully oxidized to CO₂. Folate metabolism also generates other important metabolic products, such as glycine, which is a precursor for many biosynthetic pathways. The reactions can produce and consume redox equivalents, with the oxidation of 10-formyl-THF to CO₂ generating an NADPH equivalent. The dihydrofolate-tetrahydrofolate cycle and mitochondrial-cytosolic 1C cycle are key processes in folate metabolism, with compartmentalization enabling efficient metabolic processes. Tracing studies have shown that mitochondrial-derived serine contributes significantly to homocysteine remethylation, and the deuterium tracing technique has been used to study the flux of 1C units. The impact of NADH and NADPH levels on 1C metabolism is significant, with mitochondrial 5,10-methylene-THF dehydrogenase activity linking 1C metabolism to the respiratory state of the cell. The compartmentalization of folate metabolism is encoded by different isozymes and chemical differences in folates, with the mitochondrial/cytosolic division supported by genetic and chemical factors. The role of cytosolic NADP⁺/NADPH in driving reductive cytosolic folateOne-carbon (1C) metabolism, supported by the folate cofactor, is essential for various physiological processes, including biosynthesis of purines and thymidine, amino acid homeostasis, epigenetic maintenance, and redox defense. This metabolism is compartmentalized within eukaryotic cells and across organs. The review highlights recent discoveries in mammalian 1C metabolism, emphasizing the importance of mitochondrial 1C reactions in producing 1C units for the cytosol and generating additional products like glycine and NADPH. It discusses the differential use of folate pathways in cancer, stem cells, development, and adult physiology, offering new therapeutic opportunities. Folate metabolism involves a complex set of molecules with a common core structure, including a pteridine ring, a para-aminobenzoic acid (PABA) linker, and a polyglutamate tail. The biologically active form of folate is tetrahydrofolate (THF), which carries 1C units for biosynthetic processes. 5,10-methylene-THF is used by thymidylate synthase to convert dUMP to dTMP, and by serine hydroxymethyltransferase (SHMT) to convert glycine to serine. 5-methyl-THF is involved in homocysteine remethylation to form methionine, which is crucial for S-adenosylmethionine (SAM) synthesis, a key methyl donor in epigenetics and other biosynthetic processes. 10-formyl-THF is essential for purine synthesis and can be converted to formate or fully oxidized to CO₂. Folate metabolism also generates other important metabolic products, such as glycine, which is a precursor for many biosynthetic pathways. The reactions can produce and consume redox equivalents, with the oxidation of 10-formyl-THF to CO₂ generating an NADPH equivalent. The dihydrofolate-tetrahydrofolate cycle and mitochondrial-cytosolic 1C cycle are key processes in folate metabolism, with compartmentalization enabling efficient metabolic processes. Tracing studies have shown that mitochondrial-derived serine contributes significantly to homocysteine remethylation, and the deuterium tracing technique has been used to study the flux of 1C units. The impact of NADH and NADPH levels on 1C metabolism is significant, with mitochondrial 5,10-methylene-THF dehydrogenase activity linking 1C metabolism to the respiratory state of the cell. The compartmentalization of folate metabolism is encoded by different isozymes and chemical differences in folates, with the mitochondrial/cytosolic division supported by genetic and chemical factors. The role of cytosolic NADP⁺/NADPH in driving reductive cytosolic folate