The head-to-tail dimer of the calicheamicin oligosaccharide domain exhibits substantially higher DNA binding affinity and sequence selectivity and greater bioactivity than the monomer from which it is derived. To determine the structural basis for these functional properties, the solution structure of the 1:1 complex between the head-to-tail dimer of the calicheamicin oligosaccharide and the oligonucleotide duplex d(GCACCTTCCTGC). d(GCAGGAAGGTGC) has been solved by restrained molecular dynamics calculations using NMR-derived distance and torsion angle constraints. The final input data consisted of 562 internuclear distance and 114 dihedral angle constraints, an average of 27 constraints per residue. In contrast to observations made for a complex between a DNA duplex and the head-to-head dimer of calicheamicin oligosaccharide, the head-to-tail dimer exhibits a unique binding mode in the DNA minor groove. A comparative analysis of the carbohydrate-DNA interactions at the two different binding sites explains at the atomic level how calicheamicin derivatives are able to effectively recognize both d(ACCT) and d(TCCT) sites. This study brings deeper insight into the factors governing DNA-binding affinity and the sequence preferences of calicheamicin and its derivatives.