A newly synthesized substrate, 3-hydroxybutyrylglycyl-glycyl-glycine (3HB-GGG), was applied to the assay of ACEinhibiting activity to overcome the smaller selectivity and sensitivity of the conventional method. In this study, an ACEinhibiting assay was improved by the use of a water-soluble tetrazolium salt, 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate sodium salt (WST-1), for the detection of 3-hydroxybutyrate, derived from 3HB-GGG. The optimized conditions were as follows: 0.333 mM NAD + , 0.333 mM WST-1, 0.1 mM EDTA, 0.633 U ml -1 diaphorase, and 0.700 U ml -1 3-hydroxybutyrate dehydrogenase. The developed assay was efficiently applicable to evaluate the ACEinhibiting activity of practical ACE inhibitors.