After developing a rapid gel filtration method to prepare pure and stable apoenzyme forms of D-amino acid oxidase from the yeast Rhodotorula gracilis, we carried out comparative kinetic studies on the reconstitution to holoenzyme (with FAD) of the intact (40 kDa) and proteolyzed (38.3 kDa) apoenzyme forms of this oxidase. Changes in catalytic activity and flavin and protein fluorescence revealed that in both cases reconstitution was biphasic. The proteolyzed enzyme was catalytically competent, but unlike the intact form was unable to dimerize following formation of the apoprotein-FAD complex. We present evidence that reconstitution of holoenzyme from apoenzyme plus FAD does not involve dimerization, and that dimerization is not necessary for expression of DAAO activity. We propose that both apoenzyme forms share a common reconstitution mechanism, which includes a step of conformational interconversion of an enzymatically active intermediate to the final holoenzyme.