We previously reported a novel glucose dehydrogenase (GDH) showing two peaks in the optimum temperature for the reaction at around 45 degrees C and at around 75 degrees C. Each peak derived from hetero-oligomeric enzyme, constructed from two distinct peptides with an alpha-subunit (MWs 67,000) and beta-subunit (MWs 43,000), and a single peptide enzyme containing an alpha-subunit alone. The function of the two subunits in the thermostable co-factor binding GDH was investigated. The results of spectroscopic analyses indicated that the alpha-subunit contained an unknown co-factor showing specific fluorescence spectra like pyrroloquinoline quinone (PQQ), and the beta-subunit was cytochrome c. Moreover, the results of a urea denaturation and reconstitution experiment suggested that the dissociation of the hetero-oligomeric complex to a single peptide was reversible. The kinetic parameter analyses for glucose and the electron mediator also suggested that the beta-subunit was responsible for electron transfer from the catalytic tenter of the alpha-subunit to the electron mediator.