Analysis of the genomes of 29 Escherichia coli strains revealed two different versions of the EmrE protein, a member of the small multidrug resistance family. The versions are different in length and contain 110 residues (EMRE110) and 165 residues (EMRE165). The N-terminal extension found in the longer sequence has the properties of a signal sequence, i.e. contains at the extreme N-terminus a hydrophobic region followed by a predicted cleavage site. Analysis of the genetic context of the genes in the different strains showed that all of the genes encoding EMRE165 had the same context, whereas the genes encoding EMRE110 were distributed over four different, but similar, contexts. The different genetic contexts corresponded to the branching of the phylogenetic tree of the emrE genes. Membrane topology studies using translational fusions with the two reporter proteins alkaline phosphatase and green fluorescent protein showed the well-described dual topology mode of insertion of EMRE110. In contrast, but in line with the presence of the signal sequence, EMRE165 was inserted in a single orientation into the membrane, with the C-terminus in the periplasm. The N-terminal region was removed from the protein after insertion into the membrane. In contrast to cells expressing EMRE110, cells expressing only mature EMRE165 were not able to grow on plates containing ethidium bromide. The reults suggest that if dimers were formed from EMRE165 monomers with the same orientation in the membrane, they would not be active in drug extrusion.