The first committed step in the biosynthesis of L-ascorbate from D-glucose in plants requires conversion of GDP-L-galactose to L-galactose 1-phosphate by a previously unidentified enzyme. Here we show that the protein encoded by VTC2, a gene mutated in vitamin C-deficient Arabidopsis thaliana strains, is a member of the GalT/Apa1 branch of the histidine triad protein superfamily that catalyzes the conversion of GDP-L-galactose to L-galactose 1-phosphate in a reaction that consumes inorganic phosphate and produces GDP. In characterizing recombinant VTC2 from A. thaliana as a specific GDP-L-galactose/GDP-Dglucose phosphorylase, we conclude that enzymes catalyzing each of the ten steps of the Smirnoff-Wheeler pathway from glucose to ascorbate have been identified. Finally, we identify VTC2 homologs in plants, invertebrates, and vertebrates, suggesting that a similar reaction is used widely in nature.Vitamin C (L-ascorbic acid) is well known as an important antioxidant and enzyme cofactor in animals (1, 2) and in plants (3). Apparently, all plants are able to produce vitamin C and mutants completely deficient in synthesis have not been described, suggesting an essential role of ascorbate biosynthesis in these organisms (4). Although vertebrate vitamin C synthesis is restricted to one organ (liver in mammals and kidney in fish, amphibians, and reptiles) (5, 6), virtually all cells in plants can form ascorbate (4). In the few vertebrate species, such as humans, which lack ascorbate biosynthesis, loss of the pathway is compensated by dietary intake, particularly from plants.Different pathways of ascorbate synthesis have evolved in animals and plants. In animals, ascorbate is formed from UDP-D-glucuronate in a pathway involving D-glucuronate formation, reduction and lactonization of D-glucuronate to L-gulonolactone and oxidation of the latter to L-ascorbate (reviewed in Ref. 7). Deficiency of the enzyme catalyzing this last step (L-gulonolactone oxidase) is responsible for the loss of ascorbate synthesis in the vitamin C-requiring vertebrates (8). In plants, the ascorbate synthesis pathway has remained elusive until recently and alternative pathways may exist (9). The Smirnoff-Wheeler pathway (10) has garnered strong biochemical and genetic support (11-16) and appears to represent the major route to ascorbate biosynthesis. In this pathway, GDP-D-mannose, formed from D-mannose 1-phosphate, is successively converted to GDP-L-galactose, L-galactose 1-phosphate, L-galactose, L-galactono-1,4-lactone, and finally to L-ascorbate.Screens for ozone-sensitive (17) or ascorbate-deficient (18) mutants in Arabidopsis thaliana led to the identification of four loci (VTC1, VTC2, VTC3, and VTC4) involved in the maintenance of the vitamin C pool. Characterization of the vtc1 (19) and vtc4 (15) mutants, as well as biochemical studies (14), have allowed the identification of two of the enzymes required for L-ascorbic acid synthesis through the Smirnoff-Wheeler pathway. VTC1 and VTC4 encode GDP-mannose pyrophosphorylase (19) and L-Gal-1-P ...