EmrE is a small multidrug transporter from Escherichia coli that provides a unique model for the study of polytopic membrane proteins. Here, we show its synthesis in a cell-free system in a fully functional form. The detergent-solubilized protein binds substrates with high affinity and, when reconstituted into proteoliposomes, transports substrate in a Deltamicro(H)(+)-dependent fashion. Here, we used the cell-free system to study the oligomeric properties of EmrE. EmrE functions as an oligomer, but the size of the functional oligomer has not been established unequivocally. Coexpression of two plasmids in the cell-free system allowed demonstration of functional complementation and pull-down experiments confirmed that the basic functional unit is the dimer. An additional interaction between dimers has been detected by using crosslinking between unique Cys residues. This finding implies the existence of a dimer of dimers.