A new upper limit of binding site size is defined for the hairpin polyamide-DNA motif. Ten-ring hairpin polyamides containing pyrrole (Py-) and imidazole (Im) amino acids were designed for recognition of seven base pair (bp) sequences in the minor groove of DNA. The DNA binding properties of two polyamides, ImPyPyPyPy-gamma-ImPyPyPyPy-beta-Dp, and ImImPyPyPy-gamma-Im4PyPyPyPy-beta-Dp were analyzed by footprinting and affinity cleavage on a DNA fragment containing the respective match sites 5'-TGTAACA-3' and 5'-TGGAACA-3'. Quantitative footprint titrations demonstrate that ImPyPyPyPy-gamma-ImPyPyPyPy-beta-Dp binds the 7-bp match sequence 5'-TGTAACA-3' with an equilibrium association constant (K-a) of K-a = 1.2 x 10(10) M-1 and 18-fold specificity versus the single base pair mismatch sequence 5'-TGGAACA-3'. ImImPyPyPy-gamma-ImPyPyPyPy-beta-Dp differs from ImPyPyPyPy-gamma-ImPyPyPyPy-beta-Dp by a single amino acid substitution and binds its match 5'-TGGAACA-3' site with K-a = 3.6 x 10(9) M-1 and 300-fold specificity versus its corresponding single base pair mismatch sequence 5'-TGTAACA-3'. Ten-ring hairpin polyamides have binding affinities similar to those of eight-ring hairpin polyamides, These results indicate that the affinity of hairpin binding ceases to increase as the length of the polyamide subunits increases beyond four rings, analogous to the behavior of unlinked subunits. Therefore, recognition of seven base pairs by a ten-ring hairpin polyamide most likely represents an upper limit to the effective targetable site size of the hairpin polyamide-DNA motif.