The biochemical characteristics of the formation and disappearance of intercalator-induced DNA double-strand breaks (DSB) were studied in nuclei from mouse leukemia L1210 cells by using filter elution methodology [Bradley, M. O., & Kohn, K.W. (1979) Nucleic Acids Res. 7, 793-804]. The three intercalators used were 4'-(9-acridinylamino)-methanesulfon-m-anisidide (m-AMSA), 5-iminodaunorubicin (5-ID), and ellipticine. These compounds differ in that they produced predominantly DNA single-strand breaks (SSB) (m-AMSA) or predominantly DNA double-strand breaks (ellipticine) or a mixture of both SSB and DSB (5-ID) in whole cells. In isolated nuclei, each intercalator produced DSB at a frequency comparable to that which is produced in whole cells. Moreover, these DNA breaks reversed within 30 min after drug removal. It thus appeared that neither ATP nor other nucleotides were necessary for intercalator-dependent DNA nicking-closing reactions. The formation of the intercalator-induced DSB was reduced at ice temperature. Break formation was also reduced in the absence of magnesium, at a pH above 6.4 and at NaCl concentrations above 200 mM. In the presence of ATP and ATP analogues, the intercalator-induced cleavage was enhanced. These results suggest that the intercalator-induced DSB are enzymatically mediated and that the enzymes involved in these reactions can catalyze DNA double-strand cleavage and rejoining in the absence of ATP, although the occupancy of an ATP binding site might convert the enzyme to a form more reactive to intercalators. Three inhibitors of DNA topoisomerase II--novobiocin, nalidixic acid, and norfloxacin--reduced the formation of DNA strand breaks.(ABSTRACT TRUNCATED AT 250 WORDS)