The in vitro susceptibilities of 112 clinical isolates of Haemophilus ducreyi to six antimicrobial agents were determined. These isolates were obtained in Kigali, Rwanda, during three studies on genital ulcer disease performed in 1986 (18 isolates), 1988 (23 isolates), and 1991 (71 isolates). All H. ducreyi isolates were susceptible to azithromycin, ceftriaxone, ciprofloxacin, and erythromycin; all isolates obtained in 1986 were also susceptible to trimethoprim and to the combination trimethoprim-sulfamethoxazole. In contrast, 39 and 9% of the isolates obtained in 1988 and 59 and 48% of the isolates obtained in 1991 were resistant to trimethoprim (MIC, > or = 4.0 mg/liter) and trimethoprim-sulfamethoxazole (MIC, < or = 4.0/76 mg/liter), respectively. These data indicate that trimethoprim-sulfamethoxazole can no longer be recommended for use in the treatment of chancroid in Rwanda, and possibly elsewhere in Africa.