Oligodeoxyribonucleotides cross-linked by reductively activated mitomycin C (MC) were prepared and purified for the first time. The cross-linked products were structurally characterized by nucleoside and MC-nucleoside adduct analysis. Optimal conditions were established for the cross-linking reaction, resulting in high yields, typically in the 20-50% range. Nuclease digests of the cross-linked oligonucleotides yielded the same bifunctional MC-deoxyguanosine adduct as that previously isolated from DNA exposed to MC in vitro and in vivo [Tomasz et al. (1987) Science 235, 1204]. The cross-linked oligonucleotides displayed broad thermal melting profiles, greatly increased Tm, and complex circular dichroism spectra. Phosphodiester linkages at the cross-link were resistant to spleen exonuclease, nuclease P1, and TaqI and ClaI restriction endonucleases; snake venom diesterase action was uninhibited. The cross-links are stable to heat at neutral pH but are removed by treatment in hot piperidine or by the reducing agents Na2S2O4 and dithiothreitol. Mechanisms are proposed for these reactions. These studies define optimal methods for introducing mitomycin cross-links into DNA fragments at a specific site, providing a versatile tool to study the effects of the MC cross-links on DNA structure and function.