An air-stable formate dehydrogenase, an enzyme that catalyzes the oxidation of formate to CO2 was purified from a sulfate-reducing organism, Desulfovibrio desulfuricans ATCC 27774. The enzyme has a molecular mass of approximately 150 kDa (three different subunits: 88, 29 and 16 kDa) and contains three types of redox-active centers: four c-type hemes, nonheme iron arranged as two [4Fe-4S](2+/1+) centers and a molybdenum-pterin site. Selenium was also chemically detected. The enzyme specific activity is 78 units per mg of protein. Mo(V) EPR signals were observed in the native, reduced and formate-reacted states. EPR signals related to the presence of multiple low-spin hemes were also observed in the oxidized state. Upon reduction, an examination of the EPR data under appropriate conditions distinguishes two types of iron-sulfur centers, an [Fe-S] center I (g(max) = 2.050, g(med) = 1.947, g(min) = 1.896) and an [Fe-S] center II (g(max) = 2.071, g(med) = 1.926, g(min) = 1.865). Mossbauer spectroscopy confirmed the presence of four hemes in the low-spin state. The presence of two [4Fe- 4S](2+/1+) centers was confirmed, one of these displaying very small hyperfine coupling constants in the +1 oxidation state. The midpoint redox potentials of the enzyme metal centers were also estimated.