Two-dimensional (2-D) gel electrophoresis was used to separate the extracellular proteins produced by the white-rot fungus Phanerochaete chrysosporium. Solid-substrate cultures grown on red oak wood chips yielded extracellular protein preparations which were not suitable for 2-D gel analysis. However, pre-washing the wood chips with water helped decrease the amount of brown material which caused smearing on the acidic side of the isoelectric focusing gel. The 2-D gels from these wood-grown cultures revealed more than 45 protein spots. These spots were subjected to in-gel digestion with trypsin followed by either peptide fingerprint analysis by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/MS) or by liquid chromatography (LC)/MS/MS sequencing. Data from both methods were analyzed by Protein Prospector and the local P. chrysosporium annotated database. MALDI-TOF/MS only identified two proteins out of 25 analyzed. This was most likely due to problems associated with glycosylation. Protein sequencing by LC/MS/MS of the same 25 proteins resulted in identification of 16 proteins. Most of the proteins identified act on either cellulose or hemicellulose or their hydrolysis products. Thus far no lignin peroxidase, Mn peroxidase or laccases have been detected.