Abstract Previous results indicated that resistance to acetyl-CoA carboxylase-inhibiting herbicides in the wild oat biotype UM1 was not due to an insensitive form of acetyl-CoA carboxylase (ACCase). However, reanalysis of ACCase extracted under a variety of different buffer conditions indicated that resistance in this biotype is due to an altered form of the enzyme with reduced herbicide sensitivity. Under optimal conditions, ACCase from UM1 was very resistant to sethoxydim ( I 50 = 398 μ M ; R/S I 50 ratio = 105) and resistant, although to a lesser extent, to fenoxaprop, diclofop, and tralkoxydim (R/S I 50 ratios ca. 10). Use of the optimum extraction buffer for this biotype did not indicate an ACCase-based resistance mechanism for a second resistant wild oat biotype, UM33. Fenoxaprop and diclofop were metabolized at equal rates in UM33 and a susceptible biotype, indicating that enhanced herbicide metabolism was not responsible for resistance in this biotype. Further modification of the ACCase extraction buffer revealed that resistance in UM33 was also conferred by a target site alteration. These results suggest that certain ingredients in the ACCase extraction buffers can result in the apparent loss of resistance to herbicides and that the optimum buffer may vary for biotypes within a given species. The results also suggest that careful analysis of the sensitivity of ACCase to herbicides is required before concluding that resistance is not based on a target site alteration.