The complex of a minor groove binding drug mithramycin (MTR) and the self-complementary d(TAGCTAGC TA) 10-mer duplex was investigated by molecular dynamics (MD) simulations using the AMBER 7.0 suite of programs. There is one disaccharide and trisaccharide segment projecting from opposite ends of an aglycone chromophore of MTR. A MTR dimer complex (MTR)(2)Mg2+ is formed in the presence of a coordinated ion Mg2+. A NMR solution structure of two (MTR)(2)Mg2+ Complexes bound with one DNA duplex, namely, the 2:1 duplex complex, was taken as the starting structure for the MD simulation. The partial charge on each atom was calculated using the multiple-RESP fitting procedure, and all of the missing parameters in the Parm99 force field used were adapted comparably from the literature. The length of the MD simulation was 5 ns, and the binding free energy for the formation of a 1: 1 or 2: 1 duplex complex was determined from the last 4 ns of the simulation. The binding free energies were decomposed to components of the contributions from different energy types, and the changes in the helical parameters of the bound DNA duplex plus the glycosidic linkages between sugar residues of the bound MTR dimer were determined. It was found that binding of the first (MTR)(2)Mg2+ complex with the DNA duplex to form a 1: 1 duplex complex does not cause stiffening of the duplex especially in the unoccupied site of the duplex. However, the overall flexibility of the DNA duplex is reduced substantially once the second (MTR)(2)Mg2+ complex is bound with the unoccupied site to form the 2:1 duplex complex. The van der Waals interactions were found to be dominant in the central part of the DNA duplex where sugar residues from each bound (MTR)(2)Mg2+ complex were inwardly pointing and the corresponding minor groove was widened.