ChlD mutants of Escherichia coli are pleiotropic, lacking formate-nitrate reductase activity as well as formate-hydrogenlyase activity. Whole-chain formate-nitrate reductase activity, assayed with formate as the electron donor and measuring the amount of nitrite produced, was restored to wild-type levels in the mutants by addition of 10-4 M molybdate to the growth medium. Under these conditions, the activity of each of the components of the membrane-bound nitrate reductase chain increased after molybdate supplementation. In the absence of nitrate, the activities of the formate-hydrogenlyase system were also restored by molybdate. Strains deleted for the chID gene responded in a similar way to molybdate supplementation. The concentration of molybdenum in the chlD mutant cells did not differ significantly from that in the wild-type cells at either low or high concentrations of molybdate in the medium. However, the distribution of molybdenum between the soluble protein and membrane fractions differed significantly from wild type. We conclude that the chID gene product cannot be a structural component of the formate-hydrogenlyase pathway or the formate-nitrate reductase pathway, but that it must have an indirect role in processing molybdate to a form necessary for both electron transport systems. Under anaerobic growth conditions, Escherchia coli metabolizes formate by either of two pathways (Fig. 1). In the presence of nitrate, a membrane-bound formate-nitrate reductase system is induced which oxidizes formate and reduces nitrate to nitrite (9, 20, 26). In the absence of nitrate, E. coli forms the formate-hydrogenlyase system which converts formate to carbon dioxide and hydrogen (7, 17). Mutations to chlorate resistance have been shown to result in the loss of activity of the nitrate reductase system (18). Chlorate-resistant strains, designated as either chl (2, 19) or nar (25), have been mapped at five different loci. The chMA, D, and E loci are linked to gal, chlB is linked to the metE locus, and chlC is linked to tryptophan. The chlorate-resistant mutants in classes A, B, D, and E, are pleiotropic, in that they lack the formate-hydrogenlyase pathway as well as the formate-nitrate reductase pathway (6). ChlC mutants affect only the formate-nitrate