A series of four hairpin pyrrole-imidazole polyamides, ImImPy-gamma-PyPyPy-beta-Dp, PyPyPy-gamma-ImImPy-beta-Dp, AcImImPy-gamma-PyPyPy-beta-Dp, and AcPyPyPy-gamma-ImImPy-beta-Dp (Im = N-methylimidazole-2-carboxamide, Py = N-methylpyrrole-2-carboxamide, Dp = N,N-dimethylaminopropylamide, gamma = gamma-aminobutyric acid, beta = p-alanine, and Ac = acetyl), designed for recognition of 5’-(A,T)GG(A,T)(2)-3’ sequences in the minor groove of DNA were synthesized using solid phase methodology and analyzed with respect to DNA binding affinity and sequence specificity. Quantitative DNase I footprint titration experiments reveal that the optimal polyamide ImImPy-gamma-PyPyPy-beta-Dp binds a designated 5’-TGGTT-3’ match site with an equilibrium association constant of K-a = 1.0 x 10(8) M(-1) and the single base pair mismatch sites, 5’-TGTTA-3’ and 5’-GGGTA-3’, with 50-fold and 100-fold-lower affinity, respectively (10 mM Tris HCl, 10 mM KCl, 10 mM MgCl2, and 5 mM CaCl2, pH 7.0 and 22 degrees C). Polyamides of sequence composition AcImImPy-gamma-PyPyPy-beta-Dp and AcPyPyPy-gamma-ImImPy-beta-Dp, which differ only by the position of the gamma-linker, bind with similar affinities and specificities. Recognition of sequences containing contiguous G . C base pairs expands the sequence repertoire available for targeting DNA with pyrrole-imidazole polyamides.