Short-chain monodomain family comprises pairs of membrane proteins of about 200 amino acid residues each that belong to the chromate ion transporter (CHR) superfamily. The short-chain CHR homologous pair Chr3N/Chr3C from Bacillus subtilis strain 168 confers chromate resistance only when both proteins are expressed. Membrane topology of the Chr3N and Chr3C proteins was determined in Escherichia coli by the analysis of translational fusions with reporter enzymes alkaline phosphatase and β-galactosidase. Each short-chain CHR protein was found to consist of five transmembrane segments with antiparallel orientation between them. The C terminus of Chr3N is located in the cytoplasm, whereas the C terminus of Chr3C is located in the periplasm. In silico analyses suggest that this antiparallel arrangement is shared by all protein members of the short-chain CHR3 subfamily and that the two Chr3N/Chr3C proteins might carry out distinct functions for the transport of chromate.