Conjugal mating between selenate-reducing Citrobacter sp. strain JSA and Escherichia coli S17-1 bearing pSUP2021 allowed transposon mutagenesis and chromosomal transformation. Kanamycin-resistant transconjugants were obtained successfully by this method from a freshwater selenate-respiring Citrobacter sp. strain JSA. The maximum frequency of kanamycin-resistant Tn5 transconjugants was 3.6 x 10(-6) per recipient of this strain. Of these transconjugants, eight strains of selenate reduction-deficient transconjugants living by nitrate reduction were obtained in the strain JSA. Moreover, the same phenotype of deficient mutant was created by chemical mutagenesis with ethylmethanesulfonate. The results strongly indicate that selenate reducing anaerobic respiration was independent of nitrate reduction in the Citrobacter sp. isolate strain JSA.