In order to determine whether there is an orientation preference of pyrrole-imidazole (Py-lm) polyamide dimers with respect to the 5'-3' direction of the backbone in the DNA helix, equilibrium association constants (K-a) were determined for a series of six-ring hairpin polyamides which differ with respect to substitution at the N and C termini. Affinity cleaving experiments using hairpin polyamides of core sequence composition ImPyPy-gamma-PyPyPy with an EDTA . Fe(II) moiety at the C-terminus reveal a single binding orientation at each formal match site, 5'-(A,T)G(A,T)(3)-3' and 5'-(A,T)C(A,T)(3)-3'. A positive charge at the C-terminus and no substitution at the N-terminus imidazole affords the maximum binding orientation preference, calculated from K-a(5'-TGTTA-3')/K-a(5'-TCTTA-3'), with the N-terminal end of each three-ring subunit located toward the 5' side of the target DNA strand. Removal of the positive charge, rearrangement of the positive charge to the N-terminus or substitution at the N-terminal imidazole decreases the orientation preference. These results suggest that second generation design principles superimposed on the simple pairing rules can further optimize the sequence-specificity of Py-Im polyamides for double helical DNA.