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.