The preparation of four optical isomers of 3'-hydroxyhexobarbital [5-(3'-hydroxy-1'-cyclohexen-1'-yl)-1,5-dimethylbarbituric acid] is described. The absolute configuration of the four isomers were assigned as (3'S, 5 R) and (3'R, 5R) for alpha- and beta-isomers from (R)-(--)-hexobarbital, respectively. Some of these isomeric 3'-hydroxyhexobarbitals, which are formed in the reaction of hexobarbital with liver microsomal mono-oxygenase, are oxidized to 3'-oxohexobarbital [5-(3'-oxo-1'-cyclohexen-1'-yl)-1,5-dimethylbarbituric acid] by dehydrogenase localized in the soluble fraction of liver homogenates. Comparison of activities among the four isomers for 3-hydroxyhexobarbital dehydrogenase was made by use of enzymes from guinea pig and rabbit liver. It became evident that either enzyme had a quite different activity towards each optical isomer, and the configuration of the 3'-position of hydroxyhexobarbital was an important factor affecting the reactivity of enzyme; isomers with 3'S-configuration were preferentially dehydrogenated to enantiomers with 3'R-configuration. Both enzymes had the highest activity towards (3'S, 5R)-3'-(--)-hydroxy-(--)-hexobarbital, irrespective of their having specificity towards different types of substrates.