The molybdenum- and tungsten-containing formate dehydrogenases from a variety of microorganisms catalyze the reversible interconversion of formate and CO2; several, in fact, function as CO2 reductases in the reverse direction under physiological conditions. CO2 reduction catalyzed by these enzymes occurs under mild temperature and pressure rather than the elevated conditions required for current industrial processes. Given the contemporary importance of remediation of atmospheric CO2 to address global warming, there has been considerable interest in the application of these enzymes in bioreactors. Equally important, understanding the detailed means by which these biological catalysts convert CO2 to formate, a useful and easily transported feedstock chemical, might also inspire the development of a new generation of highly efficient, biomimetic synthetic catalysts. Here we have examined the ability of the FdsDABG formate dehydrogenase from Cupriavidus necator to catalyze the exchange of solvent oxygen into product CO2 during the course of formate oxidation under single-turnover conditions. Negligible incorporation of 18O is observed when the experiment is performed in H218O, indicating that bicarbonate cannot be the immediate product of the enzyme-catalyzed reaction, as previously concluded. These results, in conjunction with the observation that the reductive half-reaction exhibits mildly acid-catalyzed rather than base-catalyzed chemistry, are consistent with a reaction mechanism involving direct hydride transfer from formate to the enzymes molybdenum center, directly yielding CO2. Our results are inconsistent with any mechanism in which the initial product formed on oxidation of formate is bicarbonate.