To identify the most active curative treatment of Buruli ulcer, two regimens incorporating the use of rifampin (RIF) were compared with the use of RIF alone in a mouse footpad model of Mycobacterium ulcerans infection. Treatments began after footpad swelling from infection and continued for 12 weeks with five doses weekly of one of the following regimens: (i) 10 mg of RIF/kg alone; (ii) 10 mg of RIF/kg and 100 mg of amikacin (AMK)/kg; and (iii) 10 mg of RIF/kg, 100 mg of clarithromycin (CLR)/kg, and 50 mg of sparfloxacin (SPX)/kg. The activity of each regimen was assessed in terms of the reduction of the average lesion index and acid-fast bacillus (AFB) and CFU counts. All three regimens displayed various degrees of bactericidal activity against M. ulcerans. The ranking of bactericidal activity was found to be as follows: RIF-AMK > RIF-CLR-SPX > RIF. RIF-AMK was able to cure M. ulcerans-infected mice and prevent relapse 26 weeks after completion of treatment. To determine the impact of different rhythms of administration of RIF-AMK on the suppression of M.ulcerans growth, mice were given the RIF-AMK combination for 4 weeks but doses were administered either 5 days a week or twice or once weekly. After completion of treatment, the mice were kept under supervision for 30 additional weeks. M. ulcerans was considered to have grown in the footpad if swelling was visually observed and harvests contained more than 5 x 10(5) AFB per footpad. The proportion of mice in which growth of M. ulcerans occurred, irrespective of drug dosage, was compared with the control mice to determine the proportion of M. ulcerans killed. Each dosage of RIF-AMK was bactericidal for M. ulcerans (P < 0.001), but the effect was significantly stronger in mice treated 5 days per week. The promising results of RIF-AMK treatment in M. ulcerans-infected mice support the clinical trial that is currently in progress under World Health Organization auspices in Ghana.