In this study, the requirements for growth factors of Ketogulonigenium vulgare LMP P-20356, a 2-keto-L-gulonic acid-producing strain of particular interest for the manufacture of vitamin C, were assessed. Various growth factors were studied in order to obtain improved growth of the strain when cultured in an L-sorbose/corn steep liquor medium. Cultures grown in the presence of reduced mono- and polyglutamated folate derivatives showed a 15- to 20-fold higher biomass content than control cultures lacking these supplements, indicating that the strain has a requirement for folate. Although most folate derivatives used in this study promoted growth, the amplitude of the response varied depending on the compound used. Dihydrofolic acid was found to be the most active form, followed by 5-formyltetrahydrofolic acid, 5-methyltetrahydrofolic acid and tetrahydrofolic acid. Folic acid had no effect. The effectiveness of polyglutamated derivatives was inversely proportional to the polyglutamated chain-length of the derivative used. Our results suggest that the rate-limiting step in the utilisation of monoglutamated folates is most probably related to their transport and/or their intracellular interconversion rather than their polymerisation into polyglutamated forms (physiological forms). The industrial production of 2-keto-L-gulonic acid by K. vulgare LMP P-20356 could be improved by using media in which low-molecular-weight reduced folates are present.