The production of pigment-free pullulan by Aureobasidium pullulans, using olive oil and sucrose as carbon (C) sources, in shake flasks, was investigated. Optimum medium composition for pullulan elaboration was 80 g/L sucrose, 25 mL/L olive oil, 5 mL/L Tween-80, 10 g/L glutamic acid, and an initial pH of 5.5. Maximum pullulan concentration (51.5 g/L), productivity (8.6 g/L.d), and yield (80.3%)were achieved under these conditions after 120 h of fermentation. The principal advantage of using olive oil and sucrose simultaneously as C sources was the elimination of the inhibitory effect of high sucrose concentrations (>60 g/L) on pullulan production by the microorganism, Structural characterization by C-13-NMR, monosaccharide, and methylation analyses, and pullulanase digestion, combined with size-exclusion chromatography, confirmed the identity of pullulan and the homogeneity of the released polysaccharide in the fermentation broths. There were no significant differences in structure between pullulan samples isolated from either olive oil-supplemented media or olive oil-free media. The molecular size of pullulan from the combined olive oil-sucrose fermentation was slightly lower (1.1 x 10(6)) than that of conventional fermentation with sucrose as a single C source (1.4 x 10(6)). Lowering the initial pH of the medium resulted in increased molecular size for the released polymer, but a lower pullulan yield.