An experimental enzyme immunoassay (EIA) for detecting serum IgG antibody to Haemophilus ducreyi was developed using an ultrasonicated whole-cell antigen. The mean optical densities (OD) for sera from men with proven chancroid from Nairobi (47 patients) and Bangkok (72 patients) were significantly higher than those obtained from Nairobi men with genital ulcers not due to H. ducreyi, from Nairobi men with urethritis, from pregnant women in Nairobi, and from European men with sexually transmitted disease. When an OD of 0.500 was taken as the cutoff value, 89% and 55% of men with proven chancroid in Nairobi and Bangkok, respectively, were positive for H. ducreyi antibody, as compared with 2%-17% in the control groups. A rise in OD was observed in five of 18 patients with clinical chancroid. These results confirm the development of circulating antibodies in chancroid and suggest that this EIA may be useful for the diagnosis and epidemiological study of H. ducreyi infection.