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Three different methods were evaluated for extraction of secreted proteins from a filamentous fungus, Cryphonectria parasitica, cultured in liquid medium. Protein samples prepared with each method were subjected to SDS-PAGE and two-dimensional gel electrophoresis (2-DE). It was found that ultrafiltration was not suitable for extraction of the secreted proteins. Protein samples prepared by chloroform treatment followed by ammonium sulfate precipitation were acceptable for 2-DE separation. The modified sevage method was the best to extract secreted proteins with highest yield and best quality. About 300 protein spots could be identified on a 2-DE gel using protein samples prepared with this method from 3 day-old culture. Successful isolation of high quality secreted proteins is the pre-request for the analysis of fungal secreted proteome. The new method may be applied to other filamentous fungi, particularly the pathogenic ones.