Supercritical fluid extraction (SFE) was used to extract cordycepin and adenosine from Cordyceps kyushuensis. An orthogonal array design (OAD) test, L-9(3)(4), including pressure, temperature, the mount of modifier and the flow rate of CO2 was performed to get the optimal conditions. The process was then scaled up by 30 times with a preparative SFE system under 40 MPa. 40 degrees C, the mount of modifier (0.04 ml min(-1)) and a flow rate of CO2 (2.01 min(-1)). The preparative SFE extracts were separated and purified by highspeed counter-current chromatography (HSCCC) with a selected two-phase solvent system composed of ethyl acetate-n-butyl alcohol-water at an optimized volume ratio of 1:4:5, and the collected fractions were analyzed by high-performance liquid chromatography (HPLC). Two nucleosides were obtained and yielded 8.92 mg of cordycepin, 5.94 mg of adenosine with purities of 98.5 and 99.2% from 400 mg SFE crude extraction, in one-step separation, respectively. (C) 2008 Elsevier B.V. All rights reserved.