We have produced and characterized a murine monoclonal antibody that recognizes DNA modified with N-acetoxy-2-acetylaminofluorene. The effectiveness of competitive binding inhibitors in an ELISA indicates that this antibody binds most strongly to N-(guanin-8-yl)-2-acetylaminofluorene. It also binds to N-(guanin-8-yl)-2-aminofluorene, albeit some 20-fold less avidly. Thus the monoclonal antibody displays specificity generally similar to the polyclonal antisera elicited by other laboratories but having the advantage of stable and precisely defined specificities. We employed a biotin-streptavidin ELISA to demonstrate that the antibody can detect less than 10 fmol of N-(guanin-8-yl)-2-acetylaminofluorene. N-(guanin-8-yl)-2-acetylaminofluorene is a more effective competitive binding inhibitor of the antibody than is N-(2'-deoxyguanosin-8-yl)-2-acetylaminofluorene or calf thymus DNA modified with N-acetoxy-2-acetylaminofluorene. Thus the antibody should be most useful in quantifying the persistence of N-acetoxy-2-acetylaminofluorene adducts in DNA hydrolyzed with trifluoroacetic acid.