Pyruvate oxidase, a tetrameric enzyme consisting of 4 identical subunits, dissociates into apoenzyme monomers and free FAD when treated with acid ammonium sulfate in the presence of high concentrations of potassium bromide. Reconstitution of the native enzymatically active protein can be accomplished by incubating equimolar concentrations of apomonomers and FAD at pH 6.5. The kinetics of the reconstitution reaction have been measured by 1) enzyme activity assays, 2) spectrophotometric assays to measure FAD binding, and 3) high performance liquid chromatography analysis measuring the distribution of monomeric, dimeric, and tetrameric species during reconstitution. The kinetic analysis indicates that the second order reaction of apomonomers with FAD to form an initial monomer-FAD complex is fast. The rate-limiting step for enzymatic reactivation appears to be the folding of the polypeptide chain in the monomer-FAD complex to reconstitute the three-dimensional FAD binding site prior to subunit reassociation. The subsequent formation of native tetramers appears to proceed via an essentially irreversible dimer assembly pathway.