From in-vitro data, recommendations for dosing with fleroxacin are presented. Serum pharmacokinetics of 250, 400, 500, 800, 1000 and 1500 mg once daily dosages were simulated in bacterial cultures. The bactericidal kinetics of clinical isolates of Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus with MICs for fleroxacin similar to MIC90 or above were investigated. Bacterial populations of all strains with MICs equal to or below 2 mg/l were reduced by at least 99% by a once daily dosage of 400 mg of fleroxacin. 500 mg once per day was high enough to induce a two log reduction of P. aeruginosa MIC 4 mg/l. At a 250 mg dosing mutants with MICs four times above the MICs of the initial strains were selected. The increased concentrations of fleroxacin after multiple dosing enhanced bactericidal activity. Once daily dosing increased the initial rate of killing but reduced the extent of inactivation in comparison with twice daily dosing of the same total amount. From our in-vitro investigation a once daily dosage of 400 mg of fleroxacin should be effective against causative organisms with an MIC of up to 2 mg/l, both in the rate and extent of killing and to minimize the risk for selection of resistant mutants.