Biochemical studies of plant species susceptible to the cyclohexanedione herbicides, alloxydim, sethoxydim, and clethodim, have demonstrated that these selective grass herbicides inhibit acetyl-coenzyme A carboxylase, the second enzyme common to both fatty acid and flavonoid biosynthetic pathways. The Kis for the cyclohexanediones tested ranged from 0.02 to 1.95 micromolar, depending on the species. The enzyme isolated from broadleaf plants was much less sensitive to inhibition by these herbicides (Kis from 53 micromolar to 2.2 millimolar). These results may explain the mechanism of action of these herbicides and their selectivity for monocotyledonous species.