Haemophilus ducreyi is the etiologic agent of the genital ulcer disease chancroid. H. ducreyi makes a secreted cytolethal distending toxin (CDT) that causes cytopathic effect on cultured human keratinocytes and fibroblasts. We indentified the gene cluster responsible for production and secretion of CDT, designated cdtABC, and constructed an isogenic H. ducreyi mutant in the structural cdtC gene.The mutant lacks CDT activity. When tested in human volunteers, this mutant was able to cause pustules that were similar to its isogenic parent strain.