Previous in vitro experiments with Desulfovibrio vulgaris strain Hildenborough demonstrated that extracts containing hydrogenase and cytochrome c3 could reduce uranium(VI) to uranium(IV) with hydrogen as the electron donor. To test the involvement of these proteins in vivo, a cytochrome c3 mutant of D. desulfuricans strain G20 was assayed and found to be able to reduce U(VI) with lactate or pyruvate as the electron donor at rates about one-half of those of the wild type. With electrons from hydrogen, the rate was more severely impaired. Cytochrome c3 appears to be a part of the in vivo electron pathway to U(VI), but additional pathways from organic donors can apparently bypass this protein.