Pretreatment of beet molasses with cation exchange resin, sulphuric acid, tricalcium phosphate, potassium ferrocyanide, and ethylenediaminetetraacetic acid and disodium salt (EDTA) to increase the production of pullulan was investigated. Among the above techniques used for the removal of heavy metals, sulphuric acid treatment gave better results regarding polysaccharide concentration, polysaccharide yield, and sugar utilization. Aureobasidium pullulans grown on beet molasses produced a mixture of pullulan and other poly saccharides. The pullulan content of the crude polysaccharide was 30-35%. The addition of nutrients improved the production of polysaccharide. A maximum polysaccharide concentration (32.0 +/- 1.0 g litre(-1)) was achieved in molasses solution (70 g litre(-1) initial sugar concentration, pH 6.5-7.5) treated with sulphuric acid and supplemented with K(2)HP0(4) 0.5%, L-glutamic acid 1%, olive oil 2.5% and Tween 80 0.5%. In this case, the highest values of biomass dry weight (33.8 +/- 1.0 g litre(-1)), polysaccharide yield (63.5 +/- 2.5%), and sugar utilization (97.5 +/- 1.5%) were obtained at pH 6.5, 3.5, and 4.5-7.5, respectively.