Serodiagnosis of chancroid is limited by the cross-reactivity of Haemophilus ducreyi with Haemophilus influenzae and Haemophilus parainfluenzae. This research describes an adsorption enzyme immunoassay (EIA) that assesses the humoral immune response of North Americans and Africans to H. ducreyi. Adsorption effectively removed anti-H. influenzae and anti-H. parainfluenzae antibodies, revealing that North American control sera had no residual anti-H. ducreyi reactivity. However, African control sera still had a residual anti-H. ducreyi response. Assessment of the duration of the humoral immune response in sera from African patients with chancroid showed that the humoral antibodies persisted for up to 8 months after the diagnosis. This may explain the lack of specificity of the adsorption EIA in areas where chancroid is endemic. The detection of the humoral immune response was affected by the strain of H. ducreyi used, with indigent strains being most useful. Using H. ducreyi 35000 for Canadian sera, the sensitivity of the adsorption EIA was 100% and the specificity was 88%. For African sera, H. ducreyi strain R018 was used, and the adsorption EIA had a sensitivity of 81% and a specificity of only 23%. These data reveal that the existing humoral response in a country where chancroid is endemic differs from that in a country where it is not, and that care must be used interpreting unadsorbed humoral immune responses. The adsorption EIA approach may prove useful as an epidemiologic tool for definition of existing (past and present) levels of exposure to H. ducreyi.