Resistance of Mycobacterium tuberculosis to fluoroquinolones (FQ) results mostly from mutations in the gyrA gene. We developed a reverse hybridization-based line probe assay in which oligonucleotide probes carrying the wild-type gyrA sequence, a serine-to-threonine (S95T) polymorphism, and gyrA mutations (A90V, A90V-S95T, S91P, S91P-S95T, D94A, D94N, D94G-S95T, D94H-S95T) were immobilized on nitrocellulose strips and hybridized with digoxigenin-labeled PCR products obtained from M. tuberculosis strains. When a mutated PCR product was used, hybridization occurred to the corresponding mutated probe but not to the wild-type probe. A panel of M. tuberculosis complex strains including 19 ofloxacin-resistant (OFL-R) and 9 ofloxacin-susceptible (OFL-S) M. tuberculosis strains was studied for detection and identification of gyrA mutations by the line probe assay and nucleotide sequencing, in comparison with testing of in vitro susceptibility to FQ. Results were 100% concordant with those of nucleotide sequencing. The S95T polymorphism, which is not related to FQ resistance, was found in 5 OFL-S and 2 OFL-R strains; the other 17 OFL-R strains harbored single mutations associated with serine or threonine at codon 95. No mutations were found in the other OFL-S strains. Overall, on the basis of the MICs on solid medium, the new line probe assay correctly identified all OFL-S and 17 out of 19 (89.5%) OFL-R strains. A nested-PCR protocol was also evaluated for the assay to amplify PCR products from M. tuberculosis-spiked sputa, with a good specificity and a sensitivity of 2 ؋ 10 3 M. tuberculosis CFU per ml of sputum. (5,(9)(10)(11)29). The use of these drugs as second-line antituberculosis agents is recommended for treating multidrug-resistant (MDR) tuberculosis (TB) (3,8). As with other antimicrobial agents, the use of FQ can generate resistant mutants (5, 6, 11-13). The principal target of the FQ in M. tuberculosis is DNA gyrase, a type II topoisomerase composed of two A and two B subunits encoded by the gyrA and gyrB genes, respectively (2,11,33). Mutations in the so-called quinolone resistance-determining region (QRDR) of gyrA are the primary mechanisms of FQ resistance in M. tuberculosis. Amino acids at positions 88, 90, 91, and 94 of gyrA are those most frequently substituted in the FQ-resistant M. tuberculosis clinical isolates (1,11,12,19,22,26,27,(31)(32)(33); mutations of gyrB have rarely been reported (15). Fluoroquinolones (FQ) are antimicrobial agents with good in vitro and in vivo activities against Mycobacterium tuberculosisDespite the increasing use of these drugs in TB therapy, standard 3-week culture-based FQ susceptibility testing in solid media is not always performed (11). PCR-based techniques provide new possibilities for the rapid diagnosis of FQ resistance; however, it can be difficult to put them into practice in the mycobacteriology laboratory (7,24,33,34). To this end, we developed a reverse hybridization-based line probe assay for rapid detection of gyrA mutations in M. tuberculosis. The test was ...