Cellobiose dehydrogenase (CDH) was purified from the brown-rot fungus Coniophora puteana grown in culture containing crystalline cellulose as a carbon source. The purified enzyme gave a single band at 115 kDa on SDS-PAGE and showed a typical flavocytochrome absorption spectrum. The enzyme oxidized both cellobiose and cellooligosaccharides, but not their monomer, glucose, suggesting typical kinetic features of CDH. A cDNA encoding CDH was cloned by RT-PCR using primers designed from the consensus sequences of known CDHs from white-rot fungi. The cDNA consists of 2448 bp, including an open reading frame encoding the 18 amino acids of the putative signal peptide and the 756 amino acids of the mature protein. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) data for tryptic fragments of the purified C. puteana CDH were consistent with partial amino acid sequences of the mature protein deduced from the cloned cDNA. Moreover, the sequences contained common characteristics of CDH, i.e., two possible residues for a heme ligand (Met 64 and His 160), a flavin-binding motif, and two glucose-methanol-choline oxidoreductase motifs. This is the first cloning of CDH from a brown-rot fungus, and the results suggest structural and kinetic similarity of C. puteana CDH to white-rot fungal CDHs.