A study of the minor-groove recognition of A/T-rich DNA sites by Ni(II)-(L)-Arg-Gly-His and Ni(II)-(D)-Arg-Gly-His was carried out with a fluorescence-based binding assay, one- and two-dimensional (1 D and 2D) NMR methodologies, and molecular simulations. Fluorescence displacement titrations revealed that Ni(II)(.)(L)-Arg-Gly-His binds to A/T-rich sequences better than the (D)-Arg diastereomer, while NMR investigations revealed that both metallopeptides bind to the minor groove of an AATT core region as evidenced by an intermolecular nuclear Overhauser effect (NOE) between each metallopeptide His imidazole C4 proton and the C2 proton of adenine. Results from molecular dynamics simulations of these systems were consistent with the experimental data and indicated that the His imidazole N-H, the N-terminal peptide amine, and Arg side chains of each metallopeptide are major determinants of minor-groove recognition by functioning as H-bond donors to the O2 of thymine residues or N3 of adenine residues.