DMT1 (divalent metal ion transporter 1) is one member of a family of proton-coupled transporters that facilitate the cellular absorption of divalent metal ions. A pair of mutation-sensitive and highly conserved histidines in the sixth transmembrane domain (TM6) of DMT1 was found to be important for proton-metal ion cotransport. In the present work, we investigate the structures and locations of the peptides from TM6 of DMT1 and its H267A and H272A mutants in SDS micelles by CD and NMR methods. The circular dichroism studies show that the alpha-helix is a predominant conformation for the wildtype peptide and H267A mutant in SDS micelles, whereas the helicity is evidently decreased for H272A mutant. The pH value has little effect on the alpha-helical contents of the three peptides. The NMR studies indicate that the wildtype peptide in SDS micelles forms an "alpha-helix-extended segment-alpha-helix" structure in which the His267 locates near the central part of the extended segment, while the His272 is involved in the alpha-helical folding. Both histidines are buried in SDS micelles as evidenced by their pK(a) values. The structure of the wildtype peptide is evidently changed by the mutations of H267A and H272A. The H267A mutant forms an ordered structure consisting of an alpha-helix from the C-terminus to the central part and continuous turns in the residual part. The extended structure in the central part of the wildtype peptide is abolished by H267A mutation. The H272A mutation mainly induces unfolding of the short helix in the N-terminal side, while the short helix in the C-terminal side and unordered conformation in the central part remain. All the three peptides are embedded in SDS micelles, and the H267A mutant is inserted more deeply due to increasing hydrophobicity in the central part of the peptide. The specific "alpha-helix-extended segment-alpha-helix" structure of TM6 may have an important implication for the binding of the transporter to H(+) and metal ions and the conformation change induced by the mutations of two highly conserved histidines may be correlated to the deficiency of the transport activity of DMT1.