Hairpin polyamides containing the aromatic amino acids 3-hydroxypyrrole (Hp), pyrrole (Py), and imidazole (Im) are capable of discriminating all four Watson-Crick base pairs in the DNA minor groove according to a set of pairing rules. Equilibrium association constants for four eight-ring hairpins containing all four pairings of Hp and Py at a single common position (ImImXPy-gamma-ImYPyPy-beta-Dp, where X/Y is Py/Py, Py/Hp, Hp/Py, and Hp/Hp) were determined at four DNA sites, 5'-TGGTCA-3', 5'-TGGACA-3', 5'-TGGACA-3', and 5'-TGGGCA-3', to study the relative binding affinities of the 16 possible complexes. The protected 3-hydroxypyrrole amino acid building block, 3-methoxypyrrole, is prepared on a 50 g scale, and the solid-phase synthesis of hydroxypyrrole-imidazole-pyrrole polyamides is described. Quantitative DNase I footprint titrations demonstrate that a Py/Py pair is partially degenerate for A.T and T.A, but disfavors G.C and C.G base pairs by 53- and 17-fold, respectively. An Hp/Py pair placed opposite T.A binds at least 20-fold more tightly than when placed opposite A.T, G.C, and C.G base pairs. The Py/Hp pair selectively binds A.T with 11-fold higher affinity over T.A and with greater than or equal to 30-fold selectivity relative to G.C and C.G. An Hp/Hp pairing is disfavored opposite all four base pairs, potentially limiting certain slipped motifs available to unlinked dimers in the minor groove. This study serves to guide the design of second-generation polyamides for DNA recognition.