Anaerobic, bacterial reduction of water-soluble U(VI) complexes to the poorly soluble U(IV) mineral uraninite has been intensively studied as a strategy for in situ remediation of uranium-contaminated groundwater. A novel and potentially counteracting metabolic process, anaerobic, nitrate-dependent U(IV) oxidation, has recently been described in two bacterial species (Geobacter metallireducens and Thiobacillus denitrificans), but the underlying biochemistry and genetics are completely unknown. We report here that two diheme, c-type cytochromes (putatively c ₄ and c ₅ cytochromes) play a major role in nitrate-dependent U(IV) oxidation by T. denitrificans. Insertion mutations in each of the two genes encoding these cytochromes resulted in a greater than 50% decrease in U(IV) oxidation activity, and complementation in trans restored activity to wild-type levels. Sucrose-density-gradient ultracentrifugation confirmed that both cytochromes are membrane-associated. Insertion mutations in genes encoding other membrane-associated, c-type cytochromes did not diminish U(IV) oxidation. This is the first report of proteins involved in anaerobic U(IV) oxidation.