Brassinolide is known to be the most biologically active compound among more than 50 brassinosteroids identified to date. However, brassinolide has not been detected in rice. To determine if this is due to the lack of the brassinolide synthase function in the rice CYP85A enzyme, we performed analyses to study metabolic conversion using a yeast strain harboring the rice CYP85A1 gene. In repeated feeding tests where the substrates were used, the biosynthetic pathway progressed only up to the synthesis of castasterone, not of brassinolide. Phylogenetic analysis of the CYP85 amino acid sequences revealed that duplication of the CYP85 gene has occurred in most dicotyledonous plant genomes; further, 1 of the 2 copies of CYP85 is evolving to develop a brassinolide synthase function. However, only a single copy of this gene is found in the currently available genome sequences of graminaceous plants; this is a likely explanation for the absence of an endogenous pool of brassinolide in rice plants.