Biopbysical investigation has been carried out for the interaction of riboflavin tetraacetate and an orotic acid derivative to know the binding mechanism between dihydroorotate dehydrogenase and dihydroorotic acid which are concerned with the oxidoreduction in vivo. Infrared and nuclear magnetic resonance spectra were measured for the chloroform solution of riboflavin tetraacetate, orotic acid butyl ester and its same mixture. Orotic acid butyl ester has been found to associate with riboflavin tetraacetate. They form a strong complex with each other. The complex is an 1:2 cyclic hydrogen bonded trimer through the imino and 2-C carbonyl groups of the isoalloxazine ring and the imino groups of the orotic acid derivative.