Recent work on the physiological responses of brassinosteroid-deficient mutants has led to the designation of brassinosteroids as a new class of phytohormone. However, information on other possible roles of brassinosteroids is limited because the mutant analysis has been confined to a relatively small number of plant species. In this context, specific inhibitors of brassinosteroid biosynthesis would be valuable for investigating the roles of brassinosteroids at various stages of plant development, such as germination, leaf expansion and flowering. Data on the oxidative processes involved in brassinosteroid biosynthesis have permitted the rational design of biosynthesis inhibitors. The development of such inhibitors has confirmed the importance of brassinosteroids in broad aspects of plant growth and development, with drastic morphological changes in inhibitor-treated plants, almost identical to those found in brassinosteroid-deficient mutants. The normal phenotype of inhibitor-treated plants can be recovered by the addition of brassinolide, revealing that inhibitors can be used to complement brassinosteroid-deficient mutants in clarifying the functions of brassinosteroids.