Three-ring polyamides containing pyrrole (Py) and imidazole (Im) amino acids covalently coupled by a turn-specific gamma-aminobutyric acid linker (gamma-turn) form six-ring hairpins that recognize predetermined 5-base pair (bp) sequences in the minor groove of DNA. To determine the sequence specificity of the gamma-turn and C-terminal beta-alanine (beta-tail) amino acids, the DNA-binding properties of the hairpin polyamide ImImPy-gamma-ImPyPy-beta-Dp were analyzed by footprinting and affinity cleavage on DNA-restriction fragments containing the eight possible 5＇-ATGGCNA-3＇ and 5＇-ANGGCTA-3＇ sites (N = A, T,G or C; 5-bp hairpin site is in italics). Quantitative footprint titrations demonstrate that both the gamma-turn and beta-tail amino acids have a > 200-400-fold preference for A.T/T.A relative to G.C base pairs at these positions, Effects of the base pairs adjacent to the 5-bp hairpin-binding site were analyzed by footprinting experiments on a DNA-restriction fragment containing the eight possible 5＇-ATGGCTN-3＇ and 5＇-NTGGCTA-3＇ sites, Quantitative footprint titrations demonstrate that the turn and tail amino acids have reduced specificity (3-20-fold preference) for A.T/T.A relative to G.C base pairs at these positions. These results indicate that the turn and tail amino acids do not simply act as neutral linker residues but, in fact, are sequence-specific recognition elements with predictable DNA-binding specificity.