The biochemical basis of resistance to the acetyl-coenzyme A carboxylase(ACCase)-inhibiting herbicide diclofop-methyl was investigated in aresistant wild oat population (R1), which does not exhibit a resistantACCase. Rates of foliar uptake and translocation of[14C]-diclofop were the same in the R1 vs. susceptible (S)populations. However, the level of phytotoxic diclofop acid was always foundto be lower in the R1 vs. S plants, with a concomitant higher level (up to1.7-fold) of nontoxic polar diclofop metabolites in R1 relative to the Splants. These results indicate that a non–target-site-based mechanism ofenhanced rate of diclofop acid metabolism confers resistance in populationR1. Moreover, the high-performance liquid chromotography elution profile ofthe major diclofop metabolites in R1 is similar to that of wheat, suggestingresistance in individuals of population R1 involves a wheat-likedetoxification system mediated by cytochrome P450 monooxygenases. Inaddition, lower level of tissue diclofop acid was also observed usingnonradioactive ultra-performance liquid chromatography–mass spectrometryanalysis in resistant individuals of three other resistant wild oatpopulations (R2, R3, and R4) known to posses ACCase gene resistancemutations. These results establish that either one or at least twoindependent resistance mechanisms (target-site ACCase resistance mutationsand non–target-site enhanced rates of herbicide metabolism) can be presentin individual wild oat plants.