A useful method for isolating and recognizing Haemophilus ducreyi from chancres and buboes of male patients is presented. A total of 41 clinical isolates of H. ducreyi were recovered from 33 patients over an 8-year period, and the experience with the 15 most recent isolates is presented in detail. Chocolate agar supplemented with 1% Iso VitaleX and 5% sheep blood agar were prepared, using Trypticase soy and Mueller-Hinton Agar bases, and incubation conditions included ambient, capneic, and anaerobic environments. Mueller-Hinton agar was clearly superior over Trypticase soy agar for isolation of H. ducreyi, although there was little difference between 5% sheep blood and supplemented chocolate agar. Growth in ambient air and under anaerobiasis was poor or lacking, whereas growth in 5 to 7% CO2 was good to luxuriant. Heat-inactivated and fresh (unheated)human blood clot tubes also were used for selective isolation. Although the rates of isolation from the two types of clot tube were not significantly different, unheated clot tubes were superior to heated clot tubes because of reduced level of contaminants. Gram stain characteristics taken from blood clot tubes and solid media, cellular and colonial morphology of the bacilli, and lack of oxidase, catalase, and biochemical activity except nitrate reductase were determinant factors. The results of this study demonstrated that successful isolation of H. ducreyi can be achieved with a minimal amount of resources and expertise.