Ascorbic acid (AsA) is present at high levels in plants and is a potent antioxidant and cellular reductant. The major plant AsA biosynthetic pathway is through the intermediates D-mannose and L-galactose. Although there is ample evidence that plants respond to fluctuating environmental conditions with changes in the pool size of AsA, it is unclear how this regulation occurs. The AsA-deficient Arabidopsis thaliana mutants vtc3-1 and vtc3-2 define a locus that has been identified by positional cloning as At2g40860. Confirmation of this identification was through the study of AsA-deficient At2g40860 insertion mutants and by transgenic complementation of the AsA deficiency in vtc3-1 and vtc3-2 with wild-type At2g40860 cDNA. The very unusual VTC3 gene is predicted to encode a novel polypeptide with an N-terminal protein kinase domain tethered covalently to a C-terminal protein phosphatase type 2C domain. Homologues of this gene exist only within the Viridiplantae/Chloroplastida and the gene may therefore have arisen along with the D-mannose/L-galactose AsA biosynthetic pathway. The vtc3 mutant plants are defective in the ability to elevate the AsA pool in response to light and heat, suggestive of an important role for VTC3 in the regulation of the AsA pool size.