We have studied the kinetics of the inhibition of mitochondrial protoporphyrinogen oxidase (PPO) from liver and placenta of 3 mammalian species by the diphenyl ether herbicide acifluorfen (AF). AF competitively inhibited PPO from human liver and placenta, mouse liver and pig placenta with respect to its substrate protoporphyrinogen. In contrast, mixed-type inhibition was shown for pig liver. The differing results shown in pig liver may point to structural differences in PPO derived from different species and tissues. We have also compared the effects of AF on the function of PPO in human lymphoblasts from normal subjects and those with variegate porphyria, an inherited disorder of PPO. Competitive inhibition was shown for both and there were no significant differences in the values of Ks or Ki.