Escherichia coli can use dimethyl sulfoxide (DMSO) as an electron acceptor during anaerobic growth on the oxidizable substrate, glycerol. During growth, the DMSO is reduced to dimethyl sulfide (DMS). For the reduction of DMSO, NADH, formate, lactate, reduced benzyl viologen, reduced methyl viologen, and dithionite can serve as electron donors. The terminal reductase and the dehyrogenases linking the various electron donors to the electron transport chain were found to be membrane bound. Chlorate-resistant mutants (chl) were unable to grow and reduce DMSO. However, in the case of the chlD mutant, growth and DMSO reduction can be restored by growth in the presence of high concentrations of molybdate. Mutants of E. coli blocked in menaquinone (vitamin K2) biosynthesis-menB, menC, and menD-were unable to grow with DMSO as an electron acceptor, even though the terminal reductase is present in these mutants. Both growth and DMSO reduction could be restored in these mutants by growth in the presence of the menaquinone intermediates, o-succinylbenzoate and 1,4-dihydroxy-2-naphthoate, depending on the metabolic block of the mutant. Thus menaquinone is involved in electron transport during DMSO reduction.