O6-Alkylguanine-DNA alkyltransferase (AGT) is an important cellular defense against the mutagenic effects of DNA alkylating agents. In humans this defense can contribute to the ability of some tumors to resist the effects of chemotherapeutic agents that act through DNA alkylation. We report here studies that characterize the interaction of AGT with DNA. We show that although AGT sediments as a monomer in the absence of DNA, it binds cooperatively to single stranded deoxyribonucleotides. The stoichiometries of complexes formed with 16-, 30-, and 80-base oligodeoxyribonucleotides are 3.8 +/- 0.3, 5.3 +/- 0.2, and 8.9 +/- 0.2, respectively; the binding density decreasing from approximately 4 nt/monomer to approximately 9 nt/monomer as DNA length increases over this range. Binding competition assays show that DNA affinities depend only weakly on base composition or secondary structure, although in general G + C-rich sequences are bound with greater affinity than are A + T-rich ones and single-stranded DNA is bound with greater affinity than duplex forms. These results suggest mechanisms by which AGT may search for alkylated sites and interact with them to effect DNA repair.