Vitamin C (L-ascorbic acid) has important antioxidant and metabolic functions in both plants and animals, humans have lost the ability to synthesize it. Fresh produce is the major source of vitamin C in the human diet yet only limited information is available concerning its route(s) of synthesis in plants. In contrast, the animal vitamin C biosynthetic pathway has been elucidated since the 1960s. Two biosynthetic pathways for vitamin C in plants are presently known. The D-mannose pathway appears to be predominant in leaf tissue, but a D-galacturonic acid pathway operates in developing fruits. Our group has previously shown that transforming lettuce and tobacco with a cDNA encoding the terminal enzyme of the animal pathway, L-gulono-1,4-lactone oxidase (GLOase, EC 1.1.3.8), increased the vitamin C leaf content between 4- and 7-fold. Additionally, we found that wild-type (wt) tobacco plants had elevated vitamin C levels when fed L-gulono-1,4-lactone, the animal precursor. These data suggest that at least part of the animal pathway may be present in plants. To further investigate this possibility, wild-type and vitamin-C-deficient Arabidopsis thaliana (L.) Heynh (vtc) plants were transformed with a 35S: GLOase construct, homozygous lines were developed, and vitamin C levels were compared to those in untransformed controls. Wild-type plants transformed with the construct showed up to a 2-fold increase in vitamin C leaf content compared to controls. All five vtc mutant lines expressing GLOase had a rescued vitamin C leaf content equal or higher (up to 3-fold) than wt leaves. These data and the current knowledge about the identity of genes mutated in the vtc lines suggest that an alternative pathway is present in plants, which can bypass the deficiency of GDP-mannose production of the vtc1-1 mutant and possibly circumvent other steps in the D-mannose pathway to synthesize vitamin C.