EmrE is an Escherichia coli multidrug transport protein that confers resistance to a wide range of toxicants by active transport across the bacterial cell membrane. The highly hydrophobic polytopic integral membrane protein has been purified and studied in its full-length form by high-resolution NMR spectroscopy in a mixture of chloroform/methanol/water (6 : 6 : 1, by vol.). Full activity is maintained after reconstitution of the protein into proteoliposomes from this solvent mixture. A series of heteronuclear (1H-15N) two- and three-dimensional experiments, as well as triple resonance experiments, were applied to the 110-residue protein and led to the assignment of the 1H, 15N and a large part of the 13C backbone resonances as well as many of the sidechain resonances. A preliminary analysis of the secondary structure, based on sequential NOE connectivities, deviation of chemical shifts from random coil values and 3JNH-Hα coupling constants supports a model where the protein forms four α-helices between residues 4−26 (TM1), 32−53 (TM2), 58−76 (TM3) and 85−106 (TM4). For the residues of helices TM2 and TM3 a significant line broadening occurs due to slow conformational processes.