Cordycepin is one of the most versatile metabolites of Cordyceps militaris. When C. natal-is G81-3, the mutant obtained by a proton beam irradiation, was cultivated by liquid surface culture, cordycepin was found to crystallize in the medium due to high cordycepin concentration. Because the cordycepin crystals strongly attached to the mycelial mat, complete recovery of cordycepin was difficult. To prevent cordycepin crystallization, increase of the initial medium volume was examined to decrease the condensation rate by vaporization, in combination with decrease of the initial substrate level. Besides, addition of the water to the culture bottle was examined to cancel out the medium condensation. A 7.4 g/L of cordycepin was obtained without crystallization by the former method. The upper limit of cordycepin production, 14.3 g/L, was obtained by the latter method and this value was the highest level of all the previous reports. A 2.5-fold scale-up of the culture slightly declined the production rate, however, the amount of cordycepin production was properly 2.5 times higher than that of the small scale. In addition, differences in sensitivities to the substrates between the mutant and the wild-type strain are discussed. (c) 2013 Elsevier Ltd. All rights reserved.