The binding of cytochrome b2 core (a monomer) to flavodehydrogenase (a tetramer), both purified from Hansenula anomala flavocytochrome b2, has been studied in the presence of 2-p-toluidinylnaphthalene-6-sulfonate (TNS). The association constant of the TNS-flavodehydrogenase complex was found to be equal to 0.64 microM-1 with a stoichiometry of one TNS per tetramer. Binding of cytochrome b2 core to flavodehydrogenase was followed by monitoring changes in the TNS fluorescence. Our results indicated that the binding is cooperative, with a stoichiometry of four cytochrome b2 cores per tetramer of flavodehydrogenase.