An extra- and an intracellular product from the bacterium Haemophilus ducreyi were shown to have a cytotoxic effect on cell lines of epithelial origin, e.g., HEp-2 and HeLa cells. The cytotoxic effect appeared on cells within 24 h and resulted in cell death and morphologically changed cells. The cytotoxic activity was heat and pronase sensitive. The activity could be removed by incubation with the target cells, suggesting attachment of the agent to the cells. The cytotoxic products were secreted into the environment during exponential growth of bacteria and produced by most of the H. ducreyi strains tested. The activity was neutralized by homologous rabbit immune serum but not by the corresponding preimmune serum. The results indicate that strains of H. ducreyi produce a cytotoxin which may be of importance to the pathogenesis of chancroid.