Optical titration studies, NMR studies and studies with DNA oligonucleotides have suggested that the carcinogen 4-nitroquinoline-1-oxide [NQO] binds to DNA with marked sequence selectivity and cooperativity and that the binding affects the reactivity of enzymes with DNA. To further examine the sequence selectivity of NQO binding to DNA, we have examined the binding of NQO [at NQO/DNA bp ratios 0.1-3] to various locations on phiX174RF DNA using restriction enzyme activity assays employing seven restriction enzymes chosen to have different reaction sequences and flanking sequences. Alterations in restriction enzyme activity were observed with the restriction enzymes Alw44, BssH II, Dra I, Stu I and Xho I while no effect was observed for cleavage by Mlu I and Pst I. While the cleavage sites for the affected enzymes differ, the flanking sequences contain similar motifs [TCTT, TGATTG, TGTTG, TCCTTG, TGTTG]. This motif type is not found in the vicinity of the unaffected enzymes. This motif type was previous shown, with oligomers, to be a hot spot for NQO binding and to be a hot spot for the binding of the carcinogen N-acetoxy-N-acetyl-2-aminofluorene. The alterations in DNA cleavage [enhanced cleavage by Alw44 and inhibition by the other enzymes] provide further indication that NQO alters DNA structure upon binding. The effects of NQO binding on DNA structure were probed using E.coli topoisomerase I assays and mung bean nuclease assays. The topoisomerase I assays suggest that NQO unwinds the DNA upon binding. NQO/DNA bp ratios from 0.07 to 0.3 produced enhancement in mung bean nuclease activity. NQO/DNA bp ratios from 0.7 to 3 produced inhibited cleavage. This change in effect with concentration may indicate cooperativity in NQO binding. These assays provide further indication that NQO distorts the structure of DNA - which could lead to the changes in restriction enzyme activity. The results presented provide further insight into the modes of carcinogen attack on DNA.