Cyclopyrimorate is a highly effective bleaching herbicide discovered by Mitsui Chemicals Agro, Inc. The target site was recently reported to be homogentisate solanesyltransferase (HST) in the plastoquinone (PQ) biosynthesis pathway on the basis of the amount of intermediates in cyclopyrimorate-treated plants and in vitro HST assays. Here, the target site of cyclopyrimorate was further explored using both in vivo and in vitro experiments.Cyclopyrimorate-dependent bleaching effect on Arabidopsis thaliana was reversed by decyl PQ, suggesting that this symptom is attributable to the inhibition of PQ biosynthesis. Furthermore, homogentisate (HGA), a substrate of HST, weakly reversed the bleaching effect of cyclopyrimorate in a dose-dependent manner. We expected that the weak reversal by HGA was due to competitive inhibition by cyclopyrimorate or des-morpholinocarbonyl cyclopyrimorate (DMC), a metabolite of cyclopyrimorate in plants that exhibits higher HST-inhibitory activity as compared to cyclopyrimorate. Therefore, kinetic analysis was conducted using DMC. DMC inhibited HST competitively with respect to HGA, and was a mixed non-competitive inhibitor with respect to the other substrate, farnesyl diphosphate. Moreover, neither cyclopyrimorate nor DMC inhibited 2-methyl-6-phytyl-1,4-benzoquinone/2-methyl-6-solanesyl-1,4-benzoquinone methyltransferase, which is located downstream of HST in the PQ biosynthesis pathway.The target site of cyclopyrimorate and DMC is HST, which is a novel target site for commercial herbicides. This article is protected by copyright. All rights reserved.