Cordycepin (3'-deoxyadenosine) is one of the most versatile metabolites of Cordyceps militaris due to its broad spectrum of biological activity. In our previous study, the C. militaris mutant G81-3, which produces higher levels of cordycepin, was obtained by high-energy proton beam irradiation. In this study, the effects of adenosine on cordycepin production in a surface liquid culture of the mutant and the wild type strains were investigated. For the mutant strain, the optimum dose of adenosine yielded a 30% increase in cordycepin production; the maximum levels of production with adenosine and without adenosine were 8.6 g/l and 6.7 g/l, respectively. In contrast, the increase due to adenosine supplementation for the wild type strain was only 15% (3.1 g/l with adenosine and 2.7 g/l without adenosine). Furthermore, a repeated batch culture, an efficient production method, was carried out to eliminate the relatively long lag phase of the mutant culture. Over four cycles, both the mutant and the wild type strain maintained a production level of more than 85% of that of the initial cycle. As a result, the disadvantage of the mutant was successfully overcome, resulting in a productivity (0.48 g/(l d)) higher than that of the batch culture (0.29 g/(l d)). The productivity for cordycepin obtained in this study is the highest reported value to date, and this method could be applied to large-scale production of cordycepin at industrial levels. (C) 2010, The Society for Biotechnology, Japan. All rights reserved.