The sulfate reducing bacterium Desulfovibrio alaskensis strain G20 can grow in lactate sulfate medium with up to 4 mM uranyl acetate. In order to identify the genes that are required for the growth of strain G20 at toxic levels of uranium(VI) (U(VI)), 5,760 transposon insertion mutants were screened for U(VI) resistance defects, and 24 of them showed loss of U(VI) resistance in lactate sulfate medium with 2 mM uranyl acetate. In the 24 mutants, 23 genes were disrupted by transposon insertions, and one transposon is located in a non-coding region. In the ten mutants that were completely inhibited by 2 mM uranyl acetate, the disrupted genes are involved in DNA repair, rRNA methylation, regulation of expression and RNA polymerase renaturation. The remaining 14 mutants showed partial inhibition of growth by 2 mM U(VI), in which the disrupted genes participate in DNA repair, regulation of transcription, membrane transport, etc. In addition, none except one of these 24 mutants showed loss in its ability to reduce U(VI) to U(IV) in the washed cell test. These results altogether suggest that U(VI) toxicity mainly involves damage to nucleic acids and proteins.