Interstrand DNA cross-links are the principal cytotoxic lesions produced by chemotherapeuticbifunctional alkylating agents. Using an N4C-ethyl-N4C interstrand DNA cross-link to mimic this classof clinically important cancer chemotherapeutic agents, we have characterized the repair, structure, andflexibility of DNA that contains this cross-link in two different orientations. Plasmid DNAs in which thecytosines of single CpG or GpC steps are covalently linked were efficiently processed by repair proficientand homologous recombination deficient strains of Escherichia coli. Repair in a nucleotide excision repair(NER) deficient strain was less efficient overall and displayed a 4-fold difference between the two crosslinkorientations. Both the structure and flexibility of DNA containing these cross-links were examinedusing a combination of 1H NMR, restrained molecular dynamics simulations, and atomic force microscopy(AFM). The NMR structure of a decamer containing a CpG interstrand cross-link shows the cross-linkeasily accommodated within the duplex with no disruption of hydrogen bonding and only minorperturbations of helical parameters. In contrast, disruptions caused by the GpC cross-link producedconsiderable conformational flexibility that precluded structure determination by NMR. AFM imaging ofcross-link-containing plasmid DNA showed that the increased flexibility observed in the GpC cross-linkpersists when it is embedded into much larger DNA fragments. These differences may account for thedifferent repair efficiencies seen in NER deficient cells.