Common waterhemp seeds were collected from two Missouri soybean fields where biotypes were not controlled by acifluorfen. Plants grown from these seeds were tested for resistance to the diphenyl ether herbicides acifluorfen and lactofen. Resistance to susceptibility (R/S) ratios, calculated as the ratio of the dose required to inhibit dry weight accumulation by 50% (GR50) in resistant plants to that for susceptible plants, were 9.5 and 11 for the Meadville biotype and 28 and 44 for the Bethel biotype exposed to acifluorfen and lactofen, respectively. Electrolyte leakage assays determined that light-induced lipid peroxidation by acifluorfen was greatest on a control population (Bradford), intermediate for the Meadville biotype, and lowest for the Bethel biotype. Levels of the photodynamic pigment protoporphyrin IX (Proto) accumulating in leaf disks exposed to acifluorfen were much lower in the resistant biotypes than in the susceptible wild type, and the level of Proto accumulation was significantly correlated to the degree of membrane disruption. Although the binding of acifluorfen to protoporphyrinogen oxidase in chloroplasts may have been altered in the resistant biotypes, the molecular and biochemical factors involved in the mechanism of resistance remain to be fully characterized. However, this study establishes that the physiological basis for the evolved resistance to diphenyl ethers in common waterhemp rests on the reduction of Proto accumulation.