The role of selenium and molybdenum in the metabolism of Escherichia coli was explored by growing cells in a simple salts medium and examining the metabolic consequences of altering the concentration of molybdenum and selenium compounds in the medium. The addition of tungstate increased the molybdate deficiency of this medium, as reflected by lowered levels of enzyme systems previously recognized to require compounds of molybdenum and selenium for their formation [formate-dependent oxygen reduction, formate dehydrogenase (FDH) (EC 1.2.2.1), and nitrate reductase (EC 1.9.6.1)]. The requirement for selenium and molybdenum appears to be unique to the enzymes of formate and nitrate metabolism since molybdate- and selenite-deficient medium had no effect on the level of several dehydrogenase and oxidase systems, for which the electron donors were reduced nicotinamide adenine dinucleotide, succinate, d- or l-lactate, and glycerol. In addition, no effect was observed on the growth rate or cell yield with any carbon source tested (glucose, glycerol, dl-lactate, acetate, succinate, and l-malate) when the medium was deficient in molybdenum and selenium. dl-Selenocystine was about as effective as selenite in stimulating the formation of formate dehydrogenase, whereas dl-selenomethionine was only 1% as effective. In aerobic cells, an amount of FDH was formed such that 3,200 or 3,800 moles of formate were oxidized per min per mole of added selenium (added as dl-selenocystine or selenite, respectively).