The antituberculosis (anti-TB) drug rifampin (RIF) binds to the beta subunit of the RNA polymerase (RpoB) of Mycobacterium tuberculosis, but the bactericidal responses triggered after target interaction are not known. To evaluate whether RIF induced an oxidative burst, lysates of RIF-treated M. tuberculosis were tested for determination of reactive oxygen species (ROS) by the electron paramagnetic resonance (EPR) technique using 1-hydroxy-3-carboxy-pyrrolidine (CPH) and 5,5-dimethyl-1-pyrrolidine-N-oxide (DMPO) as spin traps. M. tuberculosis killing by RIF stimulated an increase in the rate of formation of the CPH radical (CP·). Lysate pretreatment with the O 2 · ؊ and ·OH scavengers superoxide dismutase (SOD) and thiourea (THIO), respectively, or with the metal chelator diethylene triamine pentaacetic acid (DTPA) inhibited CP· formation, arguing in favor of a metal-catalyzed ROS response. Formation of CP· did not increase following treatment of RIF-resistant strains with RIF, indicating that the ROS were induced after RpoB binding. To identify the ROS formed, lysates of RIF-treated bacilli were incubated with DMPO, a spin trap specific for ·OH and O 2 · ؊ , with or without pretreatment with SOD, catalase, THIO, or DTPA. Superoxide dismutase, catalase, and THIO decreased formation of the DMPO-OH adduct, and SOD plus DTPA completely suppressed it, suggesting that RIF activated metal-dependent O 2 · ؊ -mediated mechanisms producing ·OH inside tubercle bacilli. The finding that the metal chelator DTPA reduced the bactericidal activity of RIF supported the possibility that ·OH was generated through these mechanisms and that it participated at least in part in M. tuberculosis killing by the drug.T uberculosis (TB), an infectious disease caused by the bacillus Mycobacterium tuberculosis, remains one of the most important public health problems, particularly in low-and middle-income countries. Current treatment for TB includes administration of first-line drugs isoniazid (INH), rifampin (RIF), pyrazinamide (PZA), and ethambutol for 2 months, followed by RIF and INH for 4 months. While molecular targets of these drugs are known (1), less information has been reported on the cellular responses triggered after target interaction. In Gram-negative and -positive bacteria, it is known that bactericidal antibiotics induced a mechanism of cellular death stimulating the production of reactive oxygen species (ROS) (2-5). The most common ROS include superoxide anion (O 2 · Ϫ ), hydrogen peroxide (H 2 O 2 ), and hydroxyl radical (·OH) (6). Using the dye hydroxyphenyl fluorescein (HPF) and flow cytometric enumeration, it was found that aminoglycosides, quinolones, and beta-lactams induced ·OH in Escherichia coli and Staphylococcus aureus through pathways involving alterations in central and iron metabolism driving the Fenton reaction (2). This reaction leads to formation of ·OH through the reduction of H 2 O 2 by ferrous iron and causes damage to DNA, proteins, and lipids that ultimately results in the death of the bacterial cell. ...