A process for simultaneous efficient production of bioactive polysaccharide and ergosterol by fed-batch fermentation of medicinal mushroom Agaricus brasiliensis was developed. Effects of carbon source and initial sugar concentration on the production of extracellular polysaccharide (EPS) and ergosterol were studied at first. Sucrose was suitable for EPS production although the cells could not grow well. Glucose was beneficial for the cell growth and production of ergosterol. When the initial glucose concentration exceeded 40 g/l, the ergosterol accumulation was decreased slightly. A repeated fed-batch fermentation strategy with glucose as carbon source, which feeding of glucose was commenced to 15 g/l from 72 to 120 h at 24 h intervals when the total glucose concentration of the broth was below 15 g/l in 71 stirred bioreactor, was developed successfully to increase simultaneously EPS and ergosterol production. The maximum EPS and ergosterol production were 1.67 +/- 0.08 g/l and 2.50 +/- 0.04 mg/100 mg DW on 144 h, respectively, which were 26.9% and 17.2% higher than those by batch fermentation in the same cultivation conditions. It was the first reported to product simultaneously EPS and ergosterol in agitated fermentation of A. brasiliensis. (c) 2005 Published by Elsevier Ltd.