The antimicrobial action of metronidazole against anaerobes is thought to be by activation due to its competition of electrons with ferredoxins, an electron-transfer protein. In this investigation, the electroreduction properties of metronidazole and eight analogues were studied by a sensitive ac polarographic technique in comparison with clostridial ferredoxin. The results showed that all the metronidazole analogues had an ac reduction peak potential that was 44-122 mV less negative than that for clostridial ferredoxin. Using Clostridium pasteurianum and Trichomonas vaginalis as the test microorganisms, the antimicrobial activities of these metronidazole analogues were determined. A theoretical expression was derived to define the relationship between the antimicrobial activity of metronidazole analogues and their activation free energy for electroreduction and lipophilicity for cell permeation. Statistical analyses of the experimental data suggested that the growth inhibition of metronidazole analogues toward Cl. pasteurianum depended on their activation free energy. For the growth inhibition of T. vaginalis, the lipophilicity of metronidazole analogues was as important as the activation free energy, as expected from the theoretical model. The competitive electroreduction between metronidazole analogues and ferredoxin was also examined. The addition of various concentrations of a metronidazole analogue to a ferredoxin solution had no effect, except to reduce the electroreductivity of ferredoxin's S-Fe bondings. This effect was observed to be directly proportional to the drug concentrations added. Thus, it was concluded that an active metronidazole analogue requires an electroreduction potential less negative than ferredoxin to be a better electron acceptor and a lower activation free energy of proton transfer to be irreversibly reduced itself to a polar derivative. This reduced species may subsequently interfere with the metabolic activity of the anaerobes, thus eliciting its antimicrobial activity.