A Lolium multiflorum Lam. biotype resistant to diclofop-methyl was found in a Triticum aestivum field in France (Normandy) that had been treated for several years with diclofop-methyl. Based on plant survival evaluated 21 d after herbicide application, the biotype exhibited a high level of resistance to diclofop-methyl and moderate resistance to CGA-184927-propargil and PP-604. The resistant biotype exhibited a small increase in tolerance to haloxyfop-methyl, quizalofop-ethyl, sethoxydim, and BAS-517-H, but was controlled by recommended field rates for these herbicides. The mechanism of resistance was investigated for diclofop-methyl. There was little or no difference in diclofop-methyl absorption by leaves of resistant and susceptible biotypes measured 48 h after treatment. For both biotypes, less than 1% of absorbed radiolabel was translocated during 48 h following foliar application of 14C-diclofop-methyl. Metabolism of diclofop-methyl was not significantly altered in the resistant biotype. In both biotypes, diclofop-methyl was rapidly metabolized to diclofop acid followed by a slow rate of formation of a polar conjugate. Two multifunctional acetyl coenzyme A carboxylase isoforms (ACCase I and ACCase II) were isolated from leaf tissue of resistant and susceptible biotypes. Both isoforms exhibited a subunit molecular mass of 203 kDa. For both resistant and susceptible biotypes, ACCase I constituted approximately 80% of total ACCase activity. Graminicide concentrations required to inhibit ACCase activity by 50% (I50 values) were determined for both ACCase isoforms from resistant and susceptible biotypes. The ACCase II isoform was highly resistant to graminicides in both biotypes. In contrast, the I50 value for diclofop inhibition of ACCase I was 19-fold greater for the enzyme isolated from the resistant biotype compared with the susceptible biotype. It is concluded that diclofop resistance in the L. multiflorum biotype from Normandy is caused by the presence of a resistant form of the ACCase I isoform.