Mitomycin C (MC) forms interstrand and intrastrand cross-link adducts and monoalkylation products (monoadducts) with DNA. Each of the three types of adducts was incorporated site-specifically into both a 15-mer and a 21-mer oligodeoxyribonucleotide duplex. The adduct-containing duplexes were 32P-phosphorylated and ligated to form multimers, which were then analyzed for anomalous electrophoretic mobility by nondenaturing polyacrylamide gel electrophoresis, using the method of Koo and Crothers [(1988) Proc. Natl. Acad. Sci. U.S.A. 85, 1763-1767] in order to detect DNA curvature caused by the adducts. The intrastrand cross-link adduct was found to induce a 14.6 +/- 2.0 degrees DNA bend per lesion (minimum value) while no DNA bending was detected for either the interstrand cross-link or the monoadduct. Molecular mechanics modeling indicated that the possible origin of the bend lies in a considerable deviation from parallel of the normals to the best planes of the intrastrand cross-linked guanines, due to a shorter than normal distance between their N2 atoms forced upon them by the cross-link. The observed bending by the MC intrastrand lesion may be the cause of the increased flexibility of MC-modified DNA, localized to distinct regions, as observed in earlier work by hydrodynamic methods and electron microscopy. The MC adduct-caused DNA bend may serve as a recognition site for certain DNA-binding proteins.