The in vitro antibacterial activity of pefloxacin was evaluated against 310 gram-negative and 315 gram-positive aerobes, freshly isolated from clinical material. Reference antibiotics were: ofloxacin, ciprofloxacin, teicoplanin, vancomycin, rifampin, and methicillin for gram-positive cocci, and norfloxacin, nalidixic acid, ceftazidime, cefotaxime, piperacillin, netilmicin, gentamicin, amikacin, and aztreonam, for gram-negative bacilli. Gram-positive cocci were inhibited by 0.5 mg/l of teicoplanin, 1 mg/l of ciprofloxacin, 4 mg/l of both pefloxacin and ofloxacin, and 8 mg/l of vancomycin or rifampicin. Against gram-negative bacteria pefloxacin showed excellent activity, inhibiting 90% of all enterobacterial strains and 70% of other gram-negative aerobes at concentrations ranging between 0.03 and 4 mg/l. The same percentage of bacteria were inhibited by the other drugs at concentrations which resulted from two to fourfold higher than those of pefloxacin. MBCs and timed-kill tests indicated that this new agent is rapidly bactericidal against these isolates, and there were no significant differences in the rate of killing of both gram-positive and gram-negative bacteria. Inoculum size and pH did not change significantly the MIC values of pefloxacin.