Two hundred fifty isolates of Mycobacterium tuberculosis were evaluated for susceptibility to ciprofloxacin, ofloxacin, levofloxacin, grepafloxacin, trovafloxacin, and gemifloxacin (SB-265805). Levofloxacin, ciprofloxacin, and grepafloxacin showed the greatest activity (MIC for 90% of strains tested [MIC 90 ] 1 g/ml), although ofloxacin also showed good activity, with an MIC 90 of 2 g/ml. Trovafloxacin and gemifloxacin showed lower in vitro activity, with MIC 90 s of 64 and 8 g/ml, respectively.The increase in drug-resistant Mycobacterium tuberculosis isolates during recent years presents a therapeutic challenge to physicians selecting antimicrobial agents (2,3,4,10,12). Fluoroquinolones may have a useful role in the treatment of these infections not only because some of their derivatives, e.g., ofloxacin (11), have already been used for the treatment of pulmonary tuberculosis but also because newer derivatives are continually being developed. However, comparative in vitro susceptibility data for classic and new agents of this class against a representative number of M. tuberculosis isolates are scarce (5,13,14).In our study, we compared the activities of the fluoroquinolones ciprofloxacin, ofloxacin, levofloxacin, grepafloxacin, trovafloxacin, and the novel compound gemifloxacin (SB-265805) against 250 clinical isolates of M. tuberculosis with different levels of susceptibility to first-line antituberculosis drugs.(Part of this study was presented as a poster at the 39th Interscience Conference on Antimicrobial Agents and Chemotherapy, 1999.) The active substances of the assayed antimycobacterial agents were kindly provided as reference powders by SmithKline Beecham (Worthing, United Kingdom). Ofloxacin was obtained from Sigma Chemical Co. (St. Louis, Mo.). Agent corrections were made for purity of antimicrobials. Stock solutions of all of the fluoroquinolones were prepared at 10,000 g/ml in distilled water by adding a 0.1 M NaOH solution for dilution when necessary. Aliquots of the antituberculosis agents were frozen at Ϫ70°C until use. Staphylococcus aureus strain ATCC 29213 was used for quality control to ensure the potency of the fluoroquinolones tested.Two hundred fifty clinical isolates of M. tuberculosis from 250 tuberculosis patients were selected from our laboratory collection (1988 to 1999). Of the samples tested, 197 were of respiratory origin and 53 were of nonrespiratory origin. Testing of susceptibility to first-line antituberculosis drugs (isoniazid, rifampin, ethambutol, and streptomycin) was performed by the agar proportion method in a reference laboratory. Of the strains tested, 44 (18%) were resistant to at least one first-line antituberculosis drug (R-MTB group; 24 monodrug-resistant and 20 multidrug-resistant strains) while the rest were fully susceptible (S-MTB group). The agar proportion method was performed as recommended by the National Committee for Clinical Laboratory Standards (9). Briefly, 7H10 agar medium (Difco) was prepared from a dehydrated base as recommended by the manufact...