Folate-mediated metabolism involves enzyme-catalyzed reactions that occur in the cytoplasmic, mitochondrial, and nuclear compartments in mammalian cells. Which of the folate-dependent enzymes are expressed in these compartments depends on the stage of development, cell type, cell cycle, and whether or not the cell is transformed. Mitochondria become formate-generating organelles in cells and tissues expressing the MTHFD2 and MTHFD1L genes. The products of these nuclear genes were derived from trifunctional precursor proteins, expressing methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase, and formyltetrahydrofolate synthetase activities. The MTHFD2 protein is a bifunctional protein with dehydrogenase and cyclohydrolase activities that arose from a trifunctional precursor through the loss of the synthetase domain and a novel adaptation to NAD rather than NADP specificity for the dehydrogenase. The MTHFD1L protein retains the size of its trifunctional precursor, but through the mutation of critical residues, both the dehydrogenase and cyclohydrolase activities have been silenced. MTHFD1L is thus a monofunctional formyltetrahydrofolate synthetase. This review discusses the properties and functions of these mitochondrial proteins and their role in supporting cytosolic purine synthesis during embryonic development and in cells undergoing rapid growth.