The (R)- and (S)-isomers of 5-methylhydantoin (5-MH) and of 5-isopropylhydantoin (5-IPH) were synthesized, and incubations of the individual isomers with a rat liver dihydropyrimidinase preparation (100,000g supernatant fraction) were carried out. Only the (R)-isomer of 5-MH or 5-IPH was ring-opened by the enzyme. Reversibility of the enzymatic ring-opening reaction could be demonstrated with only the (R)-isomer of 2-methylhydantoic acid (2-MHA) or 2-isopropylhydantoic acid (2-IPHA). The results of the present investigation show that the replacement in 5-phenylhydantoin of the phenyl group with an alkyl group does not alter the stereospecificity of the hydantoin substrates in the ring-opening reaction. The results are used to form the concept that (R)-dihydrothymine, the optical isomer previously postulated as the natural substrate to the enzyme, may have a different type of binding at the active site of the enzyme.