The cytotoxic actions of several classes of antitumor DNA intercalators are thought to result from some disturbance to DNA metabolism following trapping of the nuclear enzyme DNA topoisomerase II as a covalent complex on DNA. Here we have studied topoisomerase II trapping and DNA synthesis patterns in relation to the acute cytotoxic actions of 4′-(9-acridinylamino)methanesulfon-m-anisidide (mAMSA) or mitoxantrone on SV40 transformed human fibroblasts. These two DNA intercalators differed significantly in their cytotoxic potential, mitoxantrone being 24-fold more toxic than mAMSA when assayed by the inhibition of clonogenicity. Although both drugs induced G2 delay at cytotoxic concentrations, mAMSA-treated cells recovered normal cell cycle phase distributions within 24 h of removal of drug, while mitoxantrone-treated cells continued to accumulate in G2 up to 48 h following drug treatment with evidence of complete inhibition of entry into mitosis. Compared with mAMSA, mitoxantrone showed a similar capacity to induce cleavable complexes in cellular DNA, and only a 2-fold greater ability to inhibit DNA synthesis. Within a 4-h posttreatment period, mAMSA-treated cells recovered normal rates of DNA synthesis, whereas a continued depression of DNA synthesis was observed in mitoxantrone-treated cells. The recovery patterns of DNA synthesis correlated with the rapid disappearance of mAMSA-induced complexes (<27% lesions remaining 2 h after drug removal) and the persistence of mitoxantrone-induced complexes during a 4-h posttreatment period. This difference in complex longevity was observed in other human transformed fibroblast cell lines irrespective of differences in the absolute levels of complexes induced by either agent. We suggest that the results provide evidence that DNA intercalators may differ in the forms of complexes induced and that the comparatively high cytotoxicity of mitoxantrone relates to the ability of the drug to trap topoisomerase II complexes in a form which effects a long-term inhibition of DNA replication and G2 traverse.