The sequence-specific recognition of the minor groove of DNA by pyrrole-imidazole polyamides has been extended to 9-13 base pairs (bp). Four polyamides, ImPyPy-Py-PyPyPy-Dp, ImPyPy-beta-PyPyPy-Dp, ImPyPy-beta-PyPyPy-Dp, and ImPyPy-gamma-PyPyPy-Dp (Im = N-methylimidazole, Py = N-methylpyrrole, Dp = N,N-dimethylaminopropylamide, G = glycine, beta = beta-alanine, and gamma = gamma-aminobutyric acid), were synthesized and characterized with respect to their DNA-binding affinities and specificities at sequences of composition 5’-(A,T)G(A,T)(5)C(A,T)-3’ (9 bp) and 5’-(A,T)(5)G(A,T)C(A,T)(5)-3’ (13 bp). In both sequence contexts, the beta-alanine-linked compound ImPyPy-beta-PyPyPy-Dp has the highest binding affinity of the four polyamides, binding the 9 bp site 5’-TGTTAAACA-3’ (K-a = 8 x 10(8) M(-1)) and the 13 bp site 5’-AAAAAGACAAAAA-3’ (K-a = 5 x 10(9) M(-1)) with affinities higher than the formally N-methylpyrrole-linked polyamide ImPyPy-Py-PyPyPy-Dp by factors of similar to 8 and similar to 85, respectively (10 mM Tris . HCl, 10 mM KCl, 10 mM MgCl, and 5 mM CaCl2, pH 7.0). The binding data for ImPyPy-gamma-PyPyPy-Dp, which has been shown previously to bind DNA in a "hairpin" conformation, indicates that gamma-aminobutyric acid does not effectively link polyamide subunits in an extended conformation. These results expand the binding site size targetable with pyrrole-imidazole polyamides and provide structural elements that will facilitate the design of new polyamides targeted to other DNA sequences.