Four subjects were experimentally infected with Haemophilus ducreyi. Lesions developed only at sites where live bacteria were inoculated on abraded skin. No subject developed fever, lymphadenopathy, or disseminated infection during a 3-day observation period. Two subjects who were rechallenged 2 months after initial infection also developed lesions. The amount of H. ducreyi recovered from 10 of 12 biopsies that were semiquantitatively cultured varied widely. Similar histologic features were present in initial and second infections. The epidermis contained pustules; the dermis contained an infiltrate of T cells and macrophages and reactive endothelial cells. Keratinocytes and T cells expressed HLA-DR, consistent with a delayed-type hypersensitivity response. The subjects did not mount humoral responses to bacterial proteins and to lipooligosaccharides after primary and secondary challenges. Thus, human experimental infection with H. ducreyi is well tolerated and safe. Recruitment of T cells and macrophages into chancroid lesions may partially explain the association between chancroid and human immunodeficiency virus transmission.