Cordyceps sinensis (Cs) is a well-known traditional Chinese medicine (TCM) and Cordyceps mycelia (Cm), a cultured Cordyceps, is a substitute for Cordyceps sinensis. The most important active components in them are nucleosides. A high selective, sensitive and accurate high performance liquid chromatography method with photodiode array detection (DAD) and mass spectrometric detection has been developed for simultaneous separation, identification and quantification of nucleosides in Cs and Cm using a mobile phase including (A) ammonium acetate (40 mM, pH 5.2) and (B) methanol with a gradient system on a 2.0 mm x 150 mm Shimadzu VP-ODS column. The presence of each nucleoside in Cs and Cm was ascertained by comparison of MS data, UV spectra and retention time with standards. LC/ESI-MS in selective ion monitoring (SIM) mode were used for the quantification of nucleosides in Cs and Cm. 2-Chloroadenosine was used as internal standard for this assay. The precisions and accuracies were in the range of 1.5-5.3% and -3.5 to 5.0%, respectively. The limits of detection and quantification for nucleosides were in the order of 0.1-0.6 microg ml(-1) and 0.5-2.0 microg ml(-1), respectively. The recoveries were in the range of 92.0-107.0%. With the developed method, the concentrations of nucleosides in Cs and Cm from different sources were determined. Cs, characterized with far lower concentration of adenosine and cordycepin than Cm, can be very easy to distinguish from Cm. This reliable method would be useful for the study and quality control of Cordyceps sinensis and its substitutes.