TRPM6 and TRPM7 are two novel members of the transient receptor potential (TRP) family of ion channels and share similar channel properties. In contrast to other members of the TRPM subfamily, TRPM6 and TRPM7 are permeable for a broad range of divalent cations and are tightly regulated by intracellular Mg²⁺ and Mg²⁺-ATP. They seem to be essentially involved in the regulation of intracellular Ca²⁺ and Mg²⁺ concentrations. Multiple sequence alignment of the putative pore-forming sequence of TRPMs showed conserved negatively charged amino acid residues which might be critical for these functional characteristics. In order to investigate the role of these amino acids we performed site-directed mutagenesis. A single glutamate substitution in TRPM7, resulting in the equivalent residue of the non-selective cation channel TRPM2, provoked strong changes of the permeation properties of TRPM7. In conventional whole-cell patch-clamp measurements the mutated protein almost abrogated the block by extracellular divalent cations. Conversely, the integration of a TRPM7 amino acid sequence in the corresponding region of TRPM2 reduced inward currents and altered the selectivity for divalent cations. In conclusion, we defined amino acid residues in the pore-forming region which are critically important for the permeation characteristics of TRPM channels.