The methods for increasing the production of cordycepin (3'-deoxyadenosine), which is a kind of nucleoside analogue, were investigated by a surface culture of Cordyceps militaris NBRC 9787 using liquid medium. As a result of adding various compounds such as purine biosynthesisrelated compounds, coenzymes and surfactant to the basal medium for the purpose of enhancing the cordycepin production, glycine, L-aspartic acid, L-glutamine, adenine and adenosine were shown to be effective additives. The most preferable condition of the additives was the combination of 1 g/l of adenine and 16 g/l of glycine, and the maximum cordycepin production reached 2.5 g/l, which corresponds to 4.1 times that in the basal medium, and this value was higher compared to other reports. As a more efficient method, the repeated batch operation was shown to be practicable, and the productivity by the repeated batch operation in the optimal medium reached 0.19 g/(1d), which corresponds to 5.5 times that by the batch operation in the basal medium. The nucleic acid-related compounds produced by the surface culture were also analyzed. The results indicated that about 97% of the cordycepin synthesized by C. militaris was excreted into the culture medium, and the production of cordycepin and guanine might be linked to each other. (c) 2006 Elsevier Inc. All rights reserved.