Monoclonal antibodies were obtained after fusion of mouse P3X63-AG.8.653 myeloma cells with spleen cells isolated from BALB/cCr mice immunized with denatured DNA modified by 1-nitrosopyrene reduced with sodium ascorbate (AP-d-DNA) and complexed electrostatically to methylated bovine serum albumin. Ten stable hybridoma lines have been isolated and characterized by enzyme-linked immunosorbent assay (ELISA). They all recognize 1-aminopyrene (1-AP)-modified DNA, but not free 1-nitropyrene or 1-aminopyrene. Antibody 11H2 is the most specific for AP-DNA showing no cross-reactivity with unmodified native DNA. It also recognizes 8-nitro-1-AP and 6-nitro-1-AP modified DNA. There was some low cross-reactivity with DNA modified by a benzo[a]pyrene diol epoxide and N-acetoxy-N-2-acetylaminofluorene. Competitive ELISA with antibody 11H2 reliably detected AP-DNA adducts formed when 1-nitropyrene was incubated with Salmonella typhimurium TA1538. By immunological methods, AP-DNA adducts were shown to be unstable to heat denaturation. This suggests that specific monoclonal antibodies to carcinogen-DNA adducts will be useful not only for detecting and quantitating carcinogen-DNA damage but also for probing adduct stability.