The 26S proteasome, a large multicatalytic protease complex is involved in highly selective ATP-dependent degradation of intracellular proteins in eukaryotes, typically those tagged with a polyubiquitin chain. The proteasome-mediated proteolysis plays a key role in the regulation of critical cellular processes such as transcriptional control, cell cycle progression, and stress response. Biochemical and structural properties of the 26S proteasomes described in this paper concern the white-rot fungus Phlebia radiata, which is interesting from the standpoint of biotechnological applications of ligninolytic enzymes. In addition, the characterization of 26S proteasornes from P radiata which belongs to the class Basidiomycetes is the first one reported in the kingdom of fungi for an organism other than yeast representing the class Ascomycetes. All distinct peptidase activities: chymotrypsin-, trypsin-, and caspase-like attributed to proteasomes were detected in P. radiata proteasome preparations. Electrophoretic mobility, subunit composition, ATP-dependence, sensitivity to proteasome-specific inhibitors, and inactivation by low concentrations of SDS demonstrated for these preparations are typical features of 26S proteasome species. Moreover, findings from in vivo blocking of proteasome function strongly support the previous interpretation that the proteasomal pathway is involved in regulation of activity of some ligninolytic enzymes under nutrient deprivation.