Studying the biosynthesis of oil compounds in Perilla will help to elucidate regulatory systems for secondary metabolites and reaction mechanisms for natural product synthesis. In this study, two types of alcohol dehydrogenases, isopiperitenol dehydrogenases 1 and 2 (ISPD1 and ISPD2), which are thought to participate the oxidation of isopiperitenol in the biosynthesis of perilla, were isolated from three pure lines of perilla. Both ISPD1 and ISPD2 oxidized isopiperitenol into isopiperitenone with an oxidized form of nicotinamide adenine dinucleotide (NAD⁺) cofactor. ISPD1 used both isopiperitenol diastereomers, whereas ISPD2 used cis -isomer as a substrate. However, only ISPD2 was isolated from piperitenone-type perilla. These results suggests that in perilla, ISPD2 is related to the biosynthesis of piperitenone, which was formed via (−)- cis -isopiperitenol.