The design of synthetic ligands that read the information stored in the DNA double helix has been along-standing gal at the interface of chemistry and biology(1-5). Cell-permeable small molecules that target predetermined DNA. sequences offer a potential approach for the regulation of gene expression(6). Oligodeoxy-nucleotides that recognize the major groove of double-helical DNA via triple-helix formation bind to a broad range of sequences with high affinity and specificity(3,4), Although oligonucleotides and their analogues have been shown to interfere with gene expression(7,8), the triple-helix approach is limited to recognition of purines and suffers from poor cellular uptake. The subsequent development of pairing rules for minor-groove binding polyamides containing pyrrole (Py) and imidazole (Im) amino acids offers a second code to control sequence specificity(9-11). An Im/Py pair distinguishes G.C from C.G and both of these from A.T/T.A base pairs(9-11). A Py/Py pair specifies A.T from G.C but does not distinguish A.T from T.A(9-14). To break this degeneracy, we have added a new aromatic amino acid, 3-hydroxypyrrole (Hp), to the repertoire to test for pairings that discriminate A.T from T.A. We find that replacement of a single hydrogen atom with a hydroxy group in a Hp/Py pairing regulates affinity and specificity by an order of magnitude. By incorporation of this third amino acid, hydroxy pyrrole-imidazole-pyrrole polyamides form four ring-pairings (Im/Py, Py/Im Hp/Py and Py/Hp) which distinguish all four Watson-Crick base pairs in the minor groove of DNA.