We developed a rapid method to screen the efficacy of antimicrobial agents against Mycobacterium tuberculosis. A restriction fragment carrying a promoterless firefly luciferase gene was cloned into a 4,488-bp shuttle vector, pMV261, and luciferase was expressed under the control of a mycobacterial heat shock promoter. The resulting plasmid, pLUC10, was introduced by electroporation into the avirulent strain M. tuberculosis H37Ra. Luciferase assays of sonic lysates of Triton X-100-treated cells of M. tuberculosis H37Ra(pLUC10) yielded bioluminescence in excess of 1,000 relative light units/approximately 10(9) tubercle bacilli, compared with 0.0025 for the same number of parental cells. A 48-h microdilution antimicrobial agent-screening assay using this strain was developed.