The divalent metal transporter (DMT1) is a 12-transmembrane domain protein responsible for dietary iron uptake in the duodenum and iron acquisition from transferrin in peripheral tissues. The transmembrane domain 4 (TM4) of DMT1 has been shown to be crucial for its biological function. Here we report the 3D structure and topology of the DMT1-TM4 peptide by NMR spectroscopy with simulated annealing calculations in membrane-mimetic environments, e.g. 2,2,2-trifluoroethanol and SDS micelles. The 3D structures of the peptide are similar in both environments, with nonordered and flexible N- and C-termini flanking an ordered helical region. The final set of the 16 lowest energy structures is particularly well defined in the region of residues Leu9-Phe20 in 2,2,2-trifluoroethanol, with a mean pairwise root mean square deviation of 0.23 +/- 0.10 A for the backbone heavy atoms and 0.82 +/- 0.17 A for all heavy atoms. In SDS micelles, the length of the helix is dependent on pH values. In particular, the C-terminus becomes well-structured at low pH (4.0), whereas the N-terminal segment (Arg1-Gly7) is flexible and poorly defined at all pH values studied. The effects of 12-doxylPtdCho spin-label and paramagnetic metal ions on NMR signal intensities demonstrated that both the N-terminus and helical region of the TM4 are embedded into the interior of SDS micelles. Unexpectedly, we observed that amide protons exchanged much faster in SDS than in 2,2,2-trifluoroethanol, indicating that there is possible solvent accessibility in the structure. The paramagnetic metal ions broaden NMR signals from residues both situated in aqueous phase and in the helical region. From these results we speculate that DMT1-TM4s may self-assemble to form a channel through which metal ions are likely to be transported. These results might provide an insight into the structure-function relationship for the integral DMT1.