The in-vitro antibacterial activity of sitafloxacin (DU-6859a) against Mycobacterium leprae was evaluated and compared with those of ofloxacin, levofloxacin, and ciprofloxacin. Two biochemical indicators (intracellular ATP and uptake of [3H]-thymidine) were used to measure the in-vitro growth of M. leprae in Dhople-Hanks (DH) medium. Sitafloxacin was found to be more potent than the other three commonly used fluoroquinolones, with the minimum inhibitory concentration (MIC) against M. leprae being 0.1875 μg/ml and the action being bactericidal. The MICs of ofloxacin, levofloxacin, and ciprofloxacin were 1.5, 0.75, and 3.0 μg/ml, respectively. Similar to ofloxacin and levofloxacin, sitafloxacin also exhibited synergistic activity when combined with either rifabutin or KRM-1648, but not with rifampin. Thus, further studies on the incorporation of sitafloxacin in multidrug therapy regimens in treating leprosy patients are suggested.