The introduction of a covalent interstrand cross-link induces changes in the intrinsic structure and deformability of theDNA helix that are recognized by elements of the DNA repair apparatus. In this context, the solution structure of the undecamerd(CGAAAT*TTTCG)2, where T* represents a N3T–butyl–N3T interstrand cross-link, was determined using molecular dynamicscalculations restrained by NOE and dihedral angle data obtained from NMR spectroscopy. The structure of this cross-linked undecamershows dramatic widening of the major groove of the B-DNA stem without disruption of Watson–Crick base pairing. Thischange in tertiary structure illustrates the cumulative effect of cooperativity in intrastrand base stacking of an A-tract of three adenines.Further, it is the direct result from the imposition of geometric angular constraints by the cross-link chain on an ApT* andT*pT steps in the segment AAAT*T. The widening of the major groove is due to the dominant contribution of base stacking to thestability of the ApT compared to the TpT step suggesting that the latter is more deformable within a DNA stem. Compared to earlierstructures of ethyl cross-linked oligonucleotides, this unique perturbation induced by the butyl moiety offers a new probe forsystematic studies of DNA repair mechanisms.