Nhp6A is an abundant non-histone chromatin-associated protein in Saccharomyces cerevisiae that contains a minor groove DNA binding motif called the HMG box. In this report, we show that Nhp6Ap binds to cisplatin intrastrand cross-links on duplex DNA with a 40-fold greater affinity than to unmodified DNA with the same sequence. Nevertheless, Nhp6Ap bound to cisplatinated DNA readily exchanges onto unmodified DNA. Phenanthroline-copper footprinting and two-dimensional NMR on complexes of wild-type and mutant Nhp6Ap with DNA were employed to probe the mode of binding to the cisplatin lesion. Recognition of the cisplatin adduct requires a surface-exposed phenylalanine on Nhp6Ap that promotes bending of DNA by inserting into the helix from the minor groove. We propose that Nhp6Ap targets the cisplatin adduct by means of intercalation by the phenylalanine and that it can bind in either orientation with respect to the DNA lesion. A methionine, which also inserts between base pairs and functions in target selection on unmodified DNA, plays no apparent role in recognition of the cisplatin lesion. Basic amino acids within the N-terminal arm of Nhp6Ap are required for high-affinity binding to the cisplatin adduct as well as to unmodified DNA. Cisplatin mediates its cytotoxicity by forming covalent adducts on DNA, and we find that Deltanhp6a/b mutants are hypersensitive to cisplatin in comparison with the wild-type strain. In contrast, Deltanhp6a/b mutants are slightly more resistant to hydrogen peroxide and ultraviolet irradiation. Therefore, Nhp6A/Bp appears to directly or indirectly function in yeast to enhance cellular resistance to cisplatin.