The structure of the echinomycin complex with the self-complementary DNA decamer d(ACGTTAACGT)(2) has been investigated in solution by proton NMR spectroscopy, Echinomycin binds as a bisintercalator at the CpG steps, An analysis of the intermolecular NOE patterns defined the position and orientation of the bound drug molecule, The terminal A . T base pairs are Hoogsteen base-paired, whereas the central four A . T base pairs are clearly Watson-Crick base-paired. Thus, the presence of the extra A . T base pairs between the binding sites appears to prevent the formation of Hoogsteen base pairs immediately adjacent to the binding site, All four central A . T base pairs are destabilized relative to those in the free DNA, Because there is no evidence for unwinding of the DNA duplex in the complex relative to the free DNA, we conclude that the hypersensitivity to DNA cleavage reagents distal to echinomycin binding sites might be due to the destabilization of DNA structure induced by the drug binding.