The activity of rifabutin (LM 427) against Mycobacterium leprae was evaluated in armadillos inoculated earlier with human-derived M. leprae. Rifabutin was administered daily at a dose of 6 mg/kg body weight/day. The effect of rifabutin on M. leprae harvested from armadillos was determined by measuring the intracellular levels of ATP (an indicator of metabolic activity) of M. leprae and also their ability to multiply in the mouse footpads and in vitro in DH medium. Within 2 weeks of initiating the treatment, ATP levels declined to 21% of the original (pre-treatment level) and these M. leprae failed to multiply in the footpads of mice as well as in the in vitro culture system. This suggests that rifabutin was able to kill all M. leprae within 2 weeks. After 8 weeks the treatment was terminated and results showed that M. leprae from the treated armadillos remained non-viable in the mouse footpad system as well as in the in vitro system, indicating bactericidal action of rifabutin. The results suggest that rifabutin can be a substitute for rifampin in the leprosy multi-drug therapy regimen.