Eight-ring hairpin polyamides which differ only by the linear arrangement of pyrrole (Py) and imidazole (Im) amino acids were designed for recognition of six base pair DNA sequences containing four contiguous G,C base pairs. The respective DNA binding properties of three polyamides, ImImPyPy-gamma-ImImPyPy-beta-Dp, ImPyImPy-gamma-ImPyImPy-beta-Dp, and ImImImIm-gamma-PyPyPyPy-beta-Dp, were analyzed by footprinting and affinity cleavage on a DNA fragment containing the respective match sites 5’-TGGCCA-3’, 5’-TGCGCA-3’, and 5’-TGGGGA-3’. Quantitative footprint titrations demonstrate that ImImPyPy-gamma-ImImPyPy-beta-Dp binds the designed match site 5’-TGGCCA-3’ with an equilibrium association constant of K-a = 1 x 10(10) M-1 and >250-fold specificity versus the mismatch sequences, 5’-TGCGCA-3’ and 5’-TGGGGA-3’. The polyamides ImPyImPy-gamma-ImPyImPy-beta-Dp and ImImImIm-gamma-PyPyPyPy-beta-Dp recognize their respective 5’-TGCGCA-3’ and 5’-TGGGGA-3’ match sites with reduced affinity relative to ImImPyPy-gamma-ImImPyPy-beta-Dp, but again with high specificity with regard to mismatch sites. These results expand the DNA sequence repertoire targeted by pyrrole-imidazole polyamides and identify sequence composition effects which will, guide further second-generation polyamide design for DNA recognition.