The anticancer antibiotics mithramycin (MTR) and chromomycin A(3) (CHR) inhibit macromolecular biosynthesis by binding reversibly to double-stranded DNA via the minor groove with GC base specificity in the presence of divalent cations such as Mg2+. They have different saccharide residues. Here, the role of the sugars in DNA recognition by the antibiotics has been investigated by use of a model oligomer, d(TAGCTAGCTA)(2), that contains two partially overlapping potential binding sites (GpC). Spectroscopic studies along with analysis of binding and thermodynamic parameters for the interaction(s) of the antibiotics with the oligomer illustrate that (MTR)(2)Mg2+ binds with a 2:1 stoichiometry in contrast to a 1:1 stoichiometry for (CHR)(2)Mg2+, in terms of ligand:duplex. Both associations are enthalpy-driven, the enthalpy change being higher in the case of (MTR)(2)Mg2+. Analysis of melting profiles of the oligomer in the absence and presence of the two ligands show that (MTR)(2)Mg2+ stabilizes the duplex. On the other hand, (CHR)(2)Mg2+ stabilizes only half of the oligomer,and destabilizes the other part. This is concluded from the appearance of two peaks in the differential melting curve, where the first peak is below the melting temperature of free oligomer and the second peak is above it. The experimental results lead to the following conclusions regarding the role of the sugars. The presence of substituents such as methoxy and acetoxy groups in the A, B, and E sugars of CHR reduces the flexibility of (CHR)(2)Mg2+ ligand. As a consequence, its association with the first binding site in the oligomer leads to partial unwinding and destabilization of the second, site. Hence, (CHR)(2)Mg2+ is unable to interact with both sites in the oligomer. In contrast, (MTR)(2)Mg2+ is more flexible. It undergoes conformational changes leading to 2:1 binding stoichiometry with the oligomer. Thus, the nature of substituents in the saccharides is a major factor leading to difference in DNA recognition properties of the antibiotics.