Treatment of human neoplastic cells with dacarbazine both inhibits DNA synthesis and induces damage in the DNA. Lysis of cells in dilute alkali and subsequent electrophoretic analysis of the isolated DNA show that the DNA of treated cells includes a high molecular weight component and a population of 2-10-kilobase single-stranded DNA fragments while untreated cells contain only high molecular weight DNA. When DNA is pulse-labeled at the beginning of the dacarbazine treatment high amounts of small DNA fragments are seen but no labeled high molecular weight DNA. Moreover the DNA fragments are not formed in cells which are treated with aphidicolin before the addition of dacarbazine. Aphidicolin is a specific inhibitor of DNA polymerase alpha, the enzyme responsible for the replicative synthesis of DNA. We conclude that dacarbazine damages DNA only in cells which are synthesizing new DNA.