Clostridium pasteurianum cell-free extracts enzymatically reduced metronidazole when coupled by hydrogenase via reduced ferredoxin. A 5 mM concentration of methyl viologen, flavin adenine dinucleotide, or flavin mononucleotide could completely replace ferredoxin (0.05 mM) in the in vitro reduction assay system, whereas 5 mM benzyl viologen was less effective. However, when these electron carriers were used at a concentration of 0.05 mM, there was a drastic loss in their abilities to couple the metronidazole reduction system compared with the comparable concentration of ferredoxin. It is not understood why these flavin coenzymes participate in this enzymatic reaction. NAD and NADP had no activity when substituted for ferredoxin in the enzyme system. Two reduced ferredoxin-linked pathways, "metronidazole reductase" and the inducible dissimilatory sulfite reductase system, when combined in a single in vitro competition experiment demonstrated a preferential flow of electrons to metronidazole away from sulfite. A proposed bactericidal mechanism for metronidazole against C. pasteurianum incorporating the above findings is discussed.