A biotype of johnsongrass cross resistant to clethodim, sethoxydim, quizalofop-P, and fluazifop-P was identified in several fields in Washington County, MS. Absorption, translocation, and metabolism studies using 14C-clethodim and acetyl-coenzyme A carboxylase (ACCase) activity assays were conducted to determine the resistance mechanism. Absorption of 14C-clethodim was higher in the resistant than the susceptible biotype 4 hours after treatment (HAT), but at 24, 48, and 72 HAT, similar levels of radioactivity were detected in both johnsongrass biotypes. Consequently, resistant plants had more radioactivity present in the treated leaves at 4 and 24 HAT. However, there was no difference between resistant and susceptible biotypes in the translocation of 14C out of the treated leaf at 4, 8, 24, 48, and 72 HAT as a percentage of total absorbed. Metabolism of clethodim was similar in the resistant and susceptible biotypes. There was no difference in the specific activity of ACCase from the susceptible and resistant johnsongrass biotypes (means of 0.221 and 0.223 nmol mg−1 protein min−1, respectively). ACCase from the susceptible biotype was sensitive to clethodim, with an I50 value of 0.29 μM clethodim. The ACCase enzyme from the resistant biotype was less sensitive, with an I50 value of 1.32 μM clethodim. The resultant R/S ratio for clethodim was 4.5. These results indicate that resistance to clethodim in this johnsongrass biotype resulted from an altered ACCase enzyme that confers resistance to clethodim.