Mitomycin C (MMC) is a genotoxic cancer chemotherapeutic agent that reacts principally at the N2 position of guanine to form one of two predominant monoadducts, or a G-G interstrand cross-link at CpG sites, or a G-G intrastrand cross-link at GpG sites. Previous studies of MMC adduction have principally used very short duplex oligonucleotides (5-15 bp) or very long native duplex DNAs. We examined the formation and structural features of the MMC CpG interstrand cross-link on a model 23 bp synthetic oligonucleotide duplex having the (upper strand) sequence 5'-ATAAATACGTATTTATTTATAAA-3'. MMC was reacted with the duplex oligonucleotide in the presence of sodium dithionite at ratios of 6 mM dithionite: 1.5 mM MMC:0.03 mM duplex. The yield of cross-link in the reaction was determined to be approximately 4.8% by denaturing gel electrophoresis, which represented approximately 75% of the total bound MMC. The cross-linked DNA was isolated to greater than 97% purity in a single step by high temperature size exclusion column chromatography. Characterization of the purified product confirmed that the complex contained exclusively the N2G-MMC-N2G cross-link at the single central CpG site. CD spectroscopy demonstrated a negative band at approximately 290-320 nm which has previously been shown to be characteristic of the MMC cross-link. The relative intensity of this band compared to those reported for shorter duplexes suggested that the majority of the duplex is in a normal B-DNA helical configuration. Base-specific chemical footprinting techniques also indicated that there were subtle but distinct structural perturbations principally within the central four to six base pairs containing and adjacent to the cross-link.