ABSTRACT Tandem paired genes encoding putative short-chain monodomain protein members of the chromate ion transporter (CHR) superfamily ( ywrB and ywrA ) were cloned from genomic DNA of Bacillus subtilis strain 168. The transcription of the paired genes, renamed chr3N and chr3C , respectively, was shown to occur via a bicistronic mRNA generated from a promoter upstream of the chr3N gene. The chr3N and chr3C genes conferred chromate resistance when expressed in Escherichia coli strain W3110. The cloned chr3N gene alone did not confer chromate resistance on E. coli , suggesting that both chr3N and chr3C genes are required for function. E. coli cells expressing paired chr3N and chr3C genes demonstrated diminished uptake of chromate compared to that by a vector-only control strain. These results suggest that short-chain CHR proteins form heterodimer transporters which efflux chromate ions from the cytoplasm.