Effects of various carbon sources and carbon/nitrogen ratios on production of a useful bioactive metabolite, cordycepin (3'-deoxyadenosine), by submerged cultivation of a Chinese traditional medicinal mushroom Cordyceps militaris were investigated in shake flasks. The carbon sources examined were lactose, sucrose, glucose, fructose, galactose, maltose and xylose, and glucose was found to be most favourable to cordycepin production, whereas cells grew best in galactose medium. The dry cell weight (DW) was increased with an increase in initial glucose concentration within the range of 25-70 g/l as investigated. The highest cordycepin production, i.e. 245.7 +/- 4.4 mg/l on day 18, was obtained in medium containing 40 g glucose/l. To enhance further the cordycepin production, the effect of carbon/nitrogen ratios was studied using central composite design and response surface analysis. The maximum cordycepin production and productivity of 345.4 +/- 8.5 mg/l and 19.2 +/- 0.5 mg/l per day were achieved in medium with optimized carbon and nitrogen sources, i.e. 42.0 g glucose/l and 15.8 g peptone/l. The information obtained is helpful for the hyperproduction of cordycepin by submerged cultivation of C. militaris on a large scale. (c) 2004 Elsevier Ltd. All rights reserved.