The binding of two hairpin polyamide ligands at adjacent sites on DNA has been studied using NMR spectroscopy. The ligands lmPyPy-gamma-PyPyPy-Gly-Dp and Ac-lmPyPy-gamma-PyPyPy-Gly-Dp were studied binding to oligomers containing one or two matched binding sites: 5'-XGTTA-3' and 5'-TAACX(N)GTTA-3', where X is G, C, or A and N = 0, 1 or 2. At these sites the C-terminal ring shows an equilibrium between normal and inverted conformations. Better binding was observed with the ligand running 5' to 3' along the contacted strand than in the opposite direction. Complexes of DNAs with two binding sites indicated that at least one spacing base pair was required, and that the identity of this base pair was not critical. Binding with 5' to 3' contact is again preferred. Demonstrated binding at adjacent sites indicates that it may be possible to engineer cooperative binding for enhanced specificity or affinity.