Ascorbate is a primary antioxidant and an essential enzyme cofactor in plants, which has an important effect on the development of plant root system. To investigate the molecular mechanisms of ascorbate accumulation during root development and reveal the key genes of the ascorbate biosynthesis and recycling pathways, the expression of 16 related genes together with ascorbate abundance were analyzed in the flesh and skin of radish (Raphanus sativus L.) fleshy root. The content of ascorbate decreased with root growth in both the flesh and skin. Expression of GDP-. d-mannose pyrophosphorylase, GDP-. d-mannose-3',5'-epimerase and d-galacturonate reductase were also decreased and correlated with ascorbate levels in the flesh. In the skin, the expression of GDP-. d-mannose pyrophosphorylase and l-galactose dehydrogenase was correlated with ascorbate levels. These results suggested that ascorbate accumulation is affected mainly by biosynthesis rather than recycling in radish root, and the l-galactose pathway may be the major biosynthetic route of ascorbate, and moreover, the salvage pathway may also contribute to ascorbate accumulation. The data suggested that GDP-. d-mannose pyrophosphorylase could play an important role in the regulation of ascorbate accumulation during radish fleshy taproot development. © 2013 Elsevier Masson SAS.