Interstrand cross-links, which are generated by chemotherapeutic treatment with bis-alkylating agents, exert their therapeutic effect by connecting the nucleobases of adjacent DNA strands together and represent some of the most threatening forms of damage suffered by genomic DNA. However, one of the reasons for treatment failure using these agents is due to enhanced repair of this DNA damage. The pursuit of understanding the repair of interstrand cross-links by repair systems has necessitated the synthesis of sufficient quantities of such damaged DNA. We report the synthesis of a site-specific interstrand cross-linked duplex containing an ethylene-bridged N (1)-2'-deoxyinosine- N (3)-thymidine base pair prepared by solution and solid-phase synthesis as a mimic for the lesion formed by the therapeutic agent 1,3-bis-(2-chloroethyl)-1-nitrosourea using both a phosphoramidite and a bis-phosphoramidite approach. UV thermal denaturation experiments revealed that this cross-linked duplex was stabilized by 52 degrees C relative to the noncross-linked control, and circular dichroism studies indicated little deviation from a B-form structure compared to a duplex that contained a G-C base pair at the same position. Molecular models of the cross-linked duplex that were geometry optimized using the AMBER forcefield also suggest that this lesion induces minimal distortion in B-form DNA. This modified oligonucleotide will be useful for studies related to the investigation of interstrand cross-linked DNA repair.