Folate-dependent pathways of one-carbon metabolism are essential for the synthesis of purines, formylmethionyl-tRNA, thymidylate, serine and methionine. These syntheses use a cellular source of one-carbon substituted, tetrahydrofolate polyglutamate derivatives which are the preferred substrates of most folate-dependent enzymes. In the last decade, there have been major advances in the folate biochemistry of animal, bacterial, fungal and plant systems. These have included the refinement of methods for folate isolation and characterization, basic work on key enzymes of folate biosynthesis and the detailed characterization of proteins that catalyze the generation and utilization of one-carbon substituted folates.