DA-125, a new antitumor agent, was compared with adriamycin, a known DNA intercalator, in terms of inhibitory mechanism of DNA replication by using replicating simian virus 40 (SV40) genome in vivo. In analyzing the SV40 DNA replication intermediates present in cells treated with DA-125, it was not observed to accumulate B-dimers of SV40 DNA which are prominent in adriamycin-treated cells. However, treatment with DA-125 induced dose-dependent formation of DNA-topoisomerase complex which is characteristic of topoisomerase poisons. In addition, DA-125 showed more efficient in inhibiting SV40 DNA replication than adriamycin. Therefore, on the basis of this observation, we suggest that DA-125, a derivative of adriamycin, inhibits DNA replication by blocking topoisomerase activity as a toposomerase poison although adriamycin blocks topoisomerase activity as a DNA intercalator.