The chapter discusses the importance of one-carbon (1C) metabolism, mediated by folate, in various physiological processes such as nucleotide synthesis, amino acid homeostasis, epigenetic maintenance, and redox defense. It highlights the compartmentalization of 1C metabolic reactions within eukaryotic cells and across organs, emphasizing recent advancements in genetics, analytical chemistry, and isotope tracing. The text reviews the pathways active in different compartments, cell types, and biological states, with a focus on mitochondrial 1C reactions and their significance in producing 1C units for cytosolic use and generating products like glycine and NADPH. The chapter also explores the differential folate pathway usage in cancer, stem cells, development, and adult physiology, suggesting new opportunities for selective therapeutic intervention. Additionally, it delves into the role of folate metabolism in neural tube defects, hematopoiesis, immune response, stem cells, and non-proliferative adult tissues, providing insights into the complex interplay between 1C metabolism and various biological processes.The chapter discusses the importance of one-carbon (1C) metabolism, mediated by folate, in various physiological processes such as nucleotide synthesis, amino acid homeostasis, epigenetic maintenance, and redox defense. It highlights the compartmentalization of 1C metabolic reactions within eukaryotic cells and across organs, emphasizing recent advancements in genetics, analytical chemistry, and isotope tracing. The text reviews the pathways active in different compartments, cell types, and biological states, with a focus on mitochondrial 1C reactions and their significance in producing 1C units for cytosolic use and generating products like glycine and NADPH. The chapter also explores the differential folate pathway usage in cancer, stem cells, development, and adult physiology, suggesting new opportunities for selective therapeutic intervention. Additionally, it delves into the role of folate metabolism in neural tube defects, hematopoiesis, immune response, stem cells, and non-proliferative adult tissues, providing insights into the complex interplay between 1C metabolism and various biological processes.