HMGB1, a highly conserved non-histone DNA-binding protein, interacts with specific DNA structural motifs such as those encountered at cisplatin damage, four-way junctions, and supercoils. The interaction of full-length HMGB1, containing two tandem HMG box domains and a C-terminal acidic tail, with cisplatin-modified DNA was investigated by hydroxyl radical footprinting and electrophoretic gel mobility shift assays. The full-length HMGB1 protein binds to DNA containing a 1,2-intrastrand d(GpG) cross-link mainly through domain A, as revealed by footprinting, with a dissociation constant K-d of 120 nM. Site-directed mutagenesis of intercalating residues in both HMG domains A and B in full-length HMGB1 further supports the conclusion that only one HMG box domain is bound to the site of cisplatin damage. Interaction of the C-terminal tail with the rest of the HMGB1 protein was examined by EDC cross-linking experiments. The acidic tail mainly interacts with domain B and linker regions rather than domain A in HMGB1. These results illuminate the respective roles of the tandem HMG boxes and the C-terminal acidic tail of HMGB1 in binding to DNA and to the major DNA adducts formed by the anticancer drug cisplatin.