Allantoin racemase was isolated from cells of Candida utilis, and purified by chromatography on columns of DEAE-cellulose and Sephadex G-100. Using this purified enzyme, the racemization of allantoin in deuterium oxide was investigated. Polarimetric and PMR spectroscopic analyses showed that racemization of allantoin by the enzyme proceeded in parrallel with release of the hydrogen atom (5-H) attached to the asymmetric carbon (C-5) of allantoin. Non-enzymic racemization of allantoin, which was examined for comparison, however, was accompanied by much less or almost no release of allantoin 5-H. This indicates that the mechanism of racemization by the enzyme differs from that of non-enzymic racemization.