The effect of anthralin and its oxidation products, anthralin-dimer and anthralin-quinone, on protein content and thymidine incorporation as well as CO2 production from glucose and glutamine has been studied in transformed human keratinocytes in vitro. Anthralin exhibited the strongest inhibition, the dimer was generally less active and the quinone inactive. Respiration and thymidine incorporation were the most sensitive cellular functions showing 50% inhibition at about 1 and 3 microM anthralin, respectively. Comparison of the inhibition kinetics of anthralin with those of antimycin A and mitomycin C showed that anthralin behaved as an inhibitor of mitochondrial function rather than of DNA replication. The biologic effects were triggered in the first minutes of exposure to the cells when anthralin became rapidly associated with the cell membranes. Labeling experiments with [14C]anthralin revealed that the manifestation of the biologic response occurring after a latency phase of some hours coincided with the accumulation of radioactivity in the intracellular particulate fraction. The cytosol remained essentially unlabeled.