Diphenyl ether (DPE) herbicides such as acifluorfen inhibit both the plant and mammalian forms of protoporphyrinogen oxidase, a heme biosynthetic enzyme. Only small amounts of protoporphyrin accumulated in primary cultures of chick embryo and rat hepatocytes treated with acifluorfen and the porphyrin precursor, 5-aminolevulinic acid. However, there was a large accumulation of the porphyrin precursor, protoporphyrinogen, which was detected after oxidation to protoporphyrin by an E. coli membrane enzyme. In contrast, conventional methods of porphyrin analysis which depend on quantitative autoxidation of protoporphyrinogen failed to detect this accumulation of protoporphyrinogen. This is the first demonstration that protoporphyrinogen can accumulate to high levels and remain stable in liver cells. In addition, we found that the effect of a protoporphyrinogen oxidase inhibitor such as acifluorfen on the regulation of heme synthesis in hepatocyte cultures differed from that of an iron chelator.