The production of pigment-free pullulan by Aureobusidium pullulans in batch and fed-batch culture was investigated. Batch culture proved to be a better fermentation system for the production of pullulan than the fed-batch culture system. A maximum polysaccharide concentration (31.3 g l(-1)), polysaccharide productivity (4.5 g l(-1) per day), and sugar utilization (100%) were obtained in batch culture. In fed-batch culture, feed medium composition influenced the kinetics of fermentation. For fed-batch culture, the highest values of pullulan concentration (24.5 g l(-1)) and pullulan productivity (3.5 g l(-1) per day) were obtained in culture grown with feeding substrate containing 50 g l(-1) sucrose and all nutrients. The molecular size of pullulan showed a decline as fermentation progressed for both fermentation systems. At the end of fermentation, the polysaccharide isolated from the fed-batch culture had a slightly higher molecular weight than that of batch culture. Structural characterization of pullulan samples (methylation and enzymic hydrolysis with pullulanase) revealed the presence of mainly alpha-(1 --> 4) ( similar to 66%) and alpha-(1 --> 6) ( similar to 31%) glucosidic linkages; however, a small amount ( < 3%) of triply linked (1,3,4-, 1,3,6-, 1,2,4- and 1,4,6-Glc p) residues were detected. The molecular homogeneity of the alcohol-precipitated polysaccharides from the fermentation broths as well as the structural features of pullulan were confirmed by C-13-NMR and pullulanase treatments followed by gel filtration chromatography of the debranched digests.