Effects of nitrogen sources on cell growth and cordycepin production by submerged cultivation of Cordyceps militaris, a traditional Chinese medicinal mushroom, were investigated. In complex medium, peptone was identified as the best nitrogen source for cordycepin biosynthesis. A study using chemically defined medium indicated that NH4+ played an important role in cordycepin biosynthesis. To enhance cordycepin production, fed-batch culture with NH4+ feeding was conducted in complex medium with peptone. By optimizing the feeding time and feeding amount of NH4+, a maximal cordycepin concentration of 420.5 +/- 15.1 mg/l was obtained, which was 70% higher than that in batch cultivation. The fed-batch cultivation process was also demonstrated in a 3.51 stirred tank bioreactor and the cordycepin production was increased from 208.8 +/- 7.6 mg/l in batch culture to 346.1 +/- 3.6 mg/l in fed-batch culture. The information is useful for large-scale efficient production of cordycepin by the mushroom cultures. (c) 2005 Elsevier Inc. All rights reserved.