Laboratory and greenhouse studies were conducted to determine the mode of action of soil- and foliar-applied UCC-C4243. Experiments demonstrated that UCC-C4243 required light for phytotoxicity, phytotoxic symptoms were similar to inhibitors of porphyrin synthesis such as acifluorfen, and UCC-C4243 potently inhibited protoporphyrinogen oxidase. Germination and emergence of field pennycress and lentil in the dark were not affected by soil-incorporated UCC-C4243 at rates more than 10 times greater than like treatments that killed all plants in the light. Soil-incorporated UCC-C4243 required light for activity and killed seedlings within 1 d after emergence; sublethal doses caused desiccation, veinal necrosis, and leaf deformation. Field pennycress and lentil were susceptible to soil-incorporated UCC-C4243 and acifluorfen in the light, but were 5 to 93 times less sensitive to the herbicides in the dark. Wheat was not affected by either herbicide in the light or dark. Injury symptoms from UCC-C4243 applied POST to redroot pigweed were similar to symptoms from diphenyl ether and bipyridinium herbicides: rapid, light-dependent chlorophyll bleaching, desiccation, and necrosis. UCC-C4243, acifluorfen-methyl, and acifluorfen acid caused light- and concentration-dependent chlorophyll bleaching and electrolyte leakage from cucumber leaf disks (I50 = 1.0, 1.8, and 4.3 μM, respectively). An inhibitor of the porphyrin synthesis pathway, 4,6-dioxoheptanoic acid, almost completely inhibited herbicide-induced electrolyte leakage. δ-Aminolevulinic acid, a tetrapyrrole precursor and stimulator of the porphyrin synthesis pathway, caused synergistic effects with each herbicide. Protoporphyrinogen oxidase from barley etioplast preparations was inhibited 50% by 40 nM UCC-C4243. Barley leaf sections treated with 100 μM UCC-C4243 accumulated protoporphyrin IX in vivo to levels > 75 times non-treated controls. These data indicate the light-requiring herbicide activity of UCC-C4243, like acifluorfen, is due to inhibition of protoporphyrinogen oxidase.