An in vitro pharmacodynamic system has been successfully adapted to simulate in vivo antimicrobial pharmacokinetics under anaerobic conditions. This system was used to perform time-kill kinetic studies which were designed to compare the activity of temafloxacin to ciprofloxacin and cefotetan against two strains of Bacteroides fragilis (ATCC 25285 and ATCC 23745). All experiments were performed as single-dose, 24-h, duplicate runs. Starting bacterial inocula of 107 CFU/ml were exposed to starting antimicrobial concentrations of 5 μg of temafloxacin per ml, 5 μg of ciprofloxacin per ml, and 100 μg of cefotetan per ml. Terminal half-lives of 8, 4, and 4 h were simulated for each antimicrobial agent. Temafloxacin was rapidly bactericidal against B. fragilis. Ciprofloxacin was not bactericidal (<3 log10 unit decline in bacterial numbers) to either strain of B. fragilis. Cefotetan was bactericidal (≥3 log10 unit decline in bacterial numbers) to each strain but killed at a slower rate than temafloxacin. Times to 3 log10 unit declines of strain ATCC 25285 were 2, 4, and >24 h, whereas those of strain ATCC 23745 were 4, 4, and >24 h for temafloxacin, cefotetan, and ciprofloxacin, respectively. Total logarithmic declines of strain ATCC 25285 were >4.5, >4.5, and 2.9 log10 CFU/ml, whereas those of strain ATCC 23745 were 4.1, >4.5, and 1.2 log10 CFU/ml for each drug, respectively. These and other studies demonstrated that temafloxacin showed potential as an agent that could have been further developed for use in the treatment of anaerobic infections. However, the drug was removed from the market by its manufacturer because of toxicity issues. Although the release of newer fluoroquinolones that possess significant activity against anaerobic bacteria does not appear imminent, the time-kill studies performed in this study demonstrate that further research is warranted in the development of fluoroquinolones which possess significant antianaerobic activity.