It is known that the designed alpha-helical peptide family TRI [(Ac-G(LKALEEK)(4)G-CONH2)], containing single site substitution of a cysteine for a leucine, is capable of binding Cd(II) within a three-stranded coiled coil. The binding affinity of cadmium is dependent upon the site of substitution, with cysteine incorporated at the a site leading to cadmium complexes of higher affinity than when a d site was modified. In this work we have examined whether this differential binding affinity can be expressed in a di-cysteine-substituted peptide in order to develop site specificity within a designed system. The peptide TRI L9CL19C was used to determine whether significant differences in binding affinities at nearly proximal sites could be achieved in a short designed peptide. On the basis of Cd-113, H-1 NMR, and circular dichroic spectroscopies, we have shown that 1 equiv of Cd(II) binds exclusively at the a site. Only after that position is filled does the second site become populated. Thus, the TRI system represents the first example where stoichiometrically equivalent peptides with different sequences form the framework for designing molecular assemblies that show site-specific ion recognition. We propose that the distinct metal affinities are due to the cysteine conformers at different substitution points along the peptide. Furthermore, we have shown that site selectivity in biomolecules can be encoded into relatively short peptides with helical sequences and, therefore, do not necessarily require the extensive protein scaffolds found in natural systems.