DR-3355, the S-(-)-isomer of ofloxacin, possessed generally twice higher activity than ofloxacin, and its action was bactericidal. The difference in antibacterial activity of these compounds was attributable to their inhibitory activity against DNA gyrase. DR-3355 was characterized by its higher potency against gram-positive cocci and obligate anaerobes than ofloxacin and ciprofloxacin. DR-3355 was somewhat less potent than ciprofloxacin against Enterobacteriaceae and Pseudomonas aeruginosa, but was active against organisms resistant to cefteram, ceftazidime, and amikacin. The activity of DR-3355 was decreased by gyrA mutation, like that of the other quinolones, although it did not alter significantly by deficiency of outer membrane protein F or lipopolysaccharide. Moderately norfloxacin-resistant isolates of Staphylococcus aureus remained susceptible to DR-3355, but not to ciprofloxacin.