Escherichia coli fumarate reductase (FRD) is a four-subunit enzyme that catalyzes the terminal step in anaerobic respiration to fumarate. The hydrophobic FrdC and FrdD subunits anchor the FrdA and FrdB catalytic subunits to the inner surface of the cytoplasmic membrane and are required for the enzyme to interact with quinones. Thirty-five single-site mutations were constructed in the FrdC and FrdD polypeptides by site-directed mutagenesis. Each mutant enzyme was characterized for its ability to catalyze quinone oxidation and reduction and to support growth of E. coli DW35 (ΔfrdABCD sdhC::kan) under selective conditions requiring functional enzyme. Replacement of FrdCE29 with Asp, Leu, Lys, or Phe had a deleterious effect both on quinol oxidase and quinone reductase activities. Substitution of FrdCH82 with Arg, Leu, Tyr, or Glu also decreased menaquinol oxidase activity, but had variable effects on the reverse reaction, the reduction of ubiquinone. Data are presented to support the hypothesis that the positive charge at FrdCH82 is required for stabilization of the quinone radical intermediate and the negative charge at FrdCE29 for deprotonation of menaquinol. Other critical amino acids identified in FrdC included Ala-32, Phe-38, Trp-86, Phe-87, and in FrdD residues Phe-57, Gln-59, Ser-60, and His- 80. The established roles of such residues in the QA and QB sites of the photosynthetic reaction center would suggest a similar type of structure operative in the FRD complex. In such a model, Glu-29, Ala-32, His-82, Trp-86 of FrdC and His-80 of FrdD are considered participants in a Q(B)-type site, and FrdD Phe-57, Gln-59, and Ser-60 components in an apolar QA-type site.