Two nucleoside derivatives containing the base analogues 3-deazaadenine and 3-methyl-2-pyridone have been prepared as analogues of dA and dT, respectively. After conversion into the appropriately protected phosphoramidites, DNA sequences were prepared with site-specifically placed analogues. When present in a duplex DNA sequence, the analogues result in the deletion of one or both of the hydrogen bonding functional groups (the N3-nitrogen of dA and the O2-carbonyl of dT) present in the minor groove. Binding by two ligands, 4',6-diamidine-2-phenyl indole (DAPI) and Hoechst 33258 in the minor groove has been probed using a variety of DNA sequences. These sequences contain a d(GAATTC)2 core with analogue nucleosides substituted for one or more of the dA and dT residues. DAPI bound strongly to any sequence that contained both O2-carbonyls of the central two dT residues. The presence of a dc3A residue did in some cases enhance binding. With one of the central O2-carbonyls deleted, the binding was noticeably reduced, and with both absent, no significant binding could be detected. Similar although less dramatic results were observed with Hoechst 33258 binding to analogue sequences.