The aim of this work was to study the nutritional requirements of Zymomonas mobilis CCT 4494 for levan production in three chemically defined media. During the optimization of the fermentative process for the production of the exopolysaccharide, different concentrations of glucose, fructose and sucrose as carbon source and yeast extract as vitamin source were tested. Variations of incubation temperature and initial pH of the medium were observed. The results showed that medium containing 20.0% sucrose and 0.5% yeast extract, with initial pH of 7.0, incubated at 30°C gave a 43.0% yield of the biopolymer.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)