The effect of dihydroxyanthraquinone (DHAQ), a new antitumor drug, on mammalian chromosome replication was investigated using simian virus 40 (SV40) as a model system. The maximum effect of inhibition on viral DNA synthesis was observed within 30-40 min after the addition of the drug. The extent of inhibition of viral DNA synthesis appeared to be directly related to the number of viral replicons which interact with DHAQ molecules in vivo. No apparent strand breakage of SV40 DNA was observed in infected cells treated with DHAQ ranging from 0.3 to 10 microM. However, strand breakage was induced upon cell lysis presumably by released nuclease. Repair of the damaged SV40 chromosomes in vitro resulted in the synthesis of completed supercoiled SV40 DNA. This repair synthesis was mostly confined to the region containing the replication origin of SV40 DNA as judged by the digestion of DNA with restriction endonucleases HindII and HindIII. Since SV40 DNA sequences close to the origin of replication are not complexed with histones to form a nucleosome structure, the results suggested that DHAQ may disturb chromosome structure by interacting preferentially to the nucleosome-free regions and causing the aberrant gene duplication and expression.