A study evaluated adduct formation of the antineoplastic agent diaziquone with DNA and nucleotides in vitro. The aziridine moieties of AZQ can be expected to interact covalently with DNA which in turn presumably elicit the antitumor activity. The authors analyzed AZQ-DNA adducts by a modified 32p-postlabeling assay involving purification of the nuclease P1-enriched labeled adducts by high-salt C18 reversed-phase TLC and separation of the eluted adducts on a PEI-cellulose layer using non-urea salt solutions. Modification of calf thymus DNA with AZQ produced two major (22% and 40%) and at least eight minor adducts. At equal concentrations of AZQ and DNA (1 microgram/ul each), peak binding was observed in about 2 h (1,926 plus or minus 378 fmol/microgram DNA) with the binding levels remaining practically unchanged through 4 h. However, incubation for 24 h resulted in over 40% decline, indicating adduct instability. AZQ was found to be highly reactive in vitro as evidenced by its substantial binding (49 plus or minus 14 fmol/microgram DNA) even at a DNA:AZQ ratio of 100:1. When incubated with mononucleotides, AZQ reacted extensively with adenine, guanine, and cytosine but only slightly with thymine.