WQ-3034 is a newly synthesized acidic fluoroquinolone. We assessed its in vitro activity against Mycobacterium tuberculosis and M. avium complex using levofloxacin (LVFX), ciprofloxacin (CPFX), sparfloxacin (SPFX), and KRM-1648 (KRM) as reference drugs. The MICs of these agents were determined by the agar dilution method with 7H11 medium. The MICs at which 50 and 90% of the test strains were inhibited (MIC(50)s, and MIC(90)s, respectively) for the test quinolones for rifampin (RMP)-susceptible M. tuberculosis strains were in the order SPFX < LVFX </= WQ-3034 </= CPFX, while those for RMP-resistant M. tuberculosis strains were in the order SPFX </= WQ-3034 </= LVFX < CPFX. The MICs of KRM for RMP-susceptible M. tuberculosis were much lower than those of the test quinolones, while the MIC(90) of KRM for RMP-resistant M. tuberculosis strains was higher than those of the quinolones. The MIC(50)s and MIC(90)s of the test drugs for M. avium were in the order KRM < SPFX < CPFX </= WQ-3034 </= LVFX, while those for M. intracellulare were in the order KRM < SPFX < WQ-3034 LVFX </= CPFX. Next, we compared the antimicrobial activities of the test drugs against M. tuberculosis organisms residing in cells of the Mono Mac 6 macrophage (Mphi)-like cell line (MM6-Mphis) and of the A-549 type II alveolar cell line (A-549 cells). When drugs were added at the concentration that achieves the maximum concentration in blood, progressive killing or inhibition of the M. tuberculosis organisms residing in MM6-Mphis and A-549 cells was observed in the order KRM > SPFX >/= LVFX > WQ-3034 > CPFX. The efficacies of all quinolones against intracellular M. tuberculosis organisms were significantly lower in A-549 cells than in MM6-Mphis. WQ-3034 at the MIC caused more marked growth inhibition of intramacrophage M. tuberculosis than did LVFX. These findings indicate that the in vitro anti-M. tuberculosis activity of WQ-3034 is greater than that of CPFX and is comparable to that of LVFX.