The in vitro activity of T-3761, a new fluoroquinolone antimicrobial agent which has an oxazine ring structure with a cyclopropyl moiety at C-10, was compared with those of other agents against 2,854 clinical isolates. T-3761 had a broad spectrum of activity and had potent activity against gram-positive and -negative bacteria. The MICs of T-3761 against 90% of the methicillin-susceptible Staphylococcus aureus, methicillinsusceptible and -resistant Staphylococcus epidermidis, and Clostridium spp. tested were 0.39 to 6.25 ,g/m!. Its activity was comparable to those of ciprofloxacin and ofloxacin and four-to eightfold greater than those of norfloxacin and fleroxacin, but its activity was two-to eightfold less than that of tosufloxacin. Some isolates of ciprofloxacin-resistant S. aureus (MIC of ciprofloxacin, .3.13 ,ug/ml) were still susceptible to T-3761 (MIC of T-3761, .0.78 ,ug/ml). The MICs of T-3761 against 90%o of the streptococci and enterococci tested were 3.13 to 100 ,ug/ml. Its activity was equal to or 2-or 4-fold greater than those of norfloxacin and fleroxacin, equal to or 2-or 4-fold less than those of ofloxacin and ciprofloxacin, and 4-to 16-fold less than that of tosufloxacin. The activity of T-3761 against gram-negative bacteria was usually fourfold greater than those of norfloxacin, ofloxacin, and fleroxacin. Many isolates which were resistant to nonfluoroquinolone agents, such as minocycline-or imipenem-resistant S. aureus, ceftazidime-resistant members of the family Enterobacteriaceae, gentamicin-or imipenem-resistant Pseudomonas aeruginosa, and ampicillin-resistant Haemophilus influenzae and Neisseria gonorrhoeae, were susceptible to T-3761. The MBCs of T-3761 were either equal to or twofold greater than the MICs. The number of viable cells decreased rapidly during incubation with T-3761 at one to four times the MIC. At a concentration of four times the MIC, the frequencies of appearance of spontaneous mutants resistant to T-3761 against S. aureus, Escherichia coli, Serratia marcescens, and P. aeruginosa were 2.2 x 10-8 to <1.2 x 10-9. The 50%o inhibitory concentrations of T-3761 for DNA gyrases isolated from E. coli and P. aeruginosa were 0.88 and 1.9 ,ug/ml, respectively.