An intracellular, soluble ferrireductase thought to be involved in the reduction of manganese dioxide by white-rot fungus Phanerochaete sordida YK-624 was purified for the first time. Two isoenzymes, NAD(P)H-dependent and NADPH-dependent, respectively, were detected by hydrophobic chromatography. The NADPH-dependent ferrireductase was purified to homogeneity by ammonium sulfate fractionation, hydrophobic interaction, gel permeation, and anion-exchange chromatography. The purified protein, which is monomeric, has a molecular mass of 35 kDa (determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and pl 5.1 (determined by isoelectric focusing). The purified protein did not use cellobiose as an electron donor. The purified protein reduced Fe(III)-nitrilotriacetate complex, Mn(III)-malonate complex, methoxy-p-benzoquinone, and cytochrome c; veratraldehyde, 2-hydroxy-1,4-naphthoquinone, phenazine methosulfate, and plumbagin could not be reduced. Particularly, the protein showed the highest reducing rate for Fe(III)-organic acid complexes, such as Fe(III)-nitrilotriacetate, among these electron acceptors.