The rate of inactivation of metronidazole in vitro was determined during the course of time-kill curves against anaerobic and aerobic bacteria in the stationary phase of growth. Metronidazole at a concentration of 10 μg/ml, as measured by bioassay, was rapidly inactivated in broth culture by susceptible anaerobic bacteria (minimum bactericidal concentration ≤ 3 μg/ml), and this correlated closely with its bactericidal activity. In contrast, the drug was neither inactivated nor had any bactericidal activity against a resistant strain of Propionibacteriumacnes (minimum bactericidal concentration > 1,500 μg/ml). Three of four aerobic bacteria also inactivated metronidazole, although at generally slower rates than the anaerobes, but this was not associated with a bactericidal effect against these organisms. The presence of aerobic bacteria in mixed cultures with Bacteroides fragilis did not, moreover, inhibit the bactericidal activity of metronidazole against the latter organism. However, the possibility still remains that, in vivo, aerobic bacteria capable of inactivating metronidazole could inhibit the action of the drug against anaerobes in mixed infections.