The sequence-specific recognition of double-helical DNA by oligonucleotide-directed triple-helix formation is limited mostly to purine tracts. Design leads that couldexpand the recognition code to all fourWatson-Crick base pairs would provide one step toward a general solution targeting single sites in megabase size DNA. The nonnatural deoxyribonucleoside l-(2-deoxy-/3-D-ribofuranosyl)-4-(3-benzamidophenyl) imidazole (D3) was synthesized infour steps and incorporated by automated methods intopyrimidine oligodeoxyribonucleotides. Within a pyrimidine oligonucleotide, D3 binds pyrimidine-purine base pairs with higher affinity than it binds purine-pyrimidine base pairs. Fromaffinity-cleaving analysis, the stabilitiesof base triplets decrease in the order D3-TA~ D3-CG> D3-AT> D3-GC. Such specificity allows binding by triple-helix formation at an 18 base pair site in SV40 DNA containing all four base pairs at physiologically relevant pH and temperature. The stabilities of these novel triplets may be an example of shape-selective recognition of CG and TA Watson-Crick base pairsin the major groove.