When electrophoresed on an agarose gel, the DNA isolated from neocarzinostatin- (NCS-) treated HeLa cells migrates in a ladder of discrete bands indicative of preferential breakage in the linker region of the nucleosomes. The 5’-termini of the drug-induced DNA strand breaks were characterized by (1) reduction of the nucleoside 5’-aldehyde ends to 5’-hydroxyls followed by incorporation of 32P from [γ-32P] ATP by polynucleotide kinase and (2) treatment of the DNA with hot alkali and alkaline phosphatase prior to the kinase assay to give the total 5’-termini. In DNA isolated from NCS-treated cells, nucleoside aldehyde accounts for 30-45% of the drug-generated 5’ ends; the remainder have PO4 termini. By contrast, 5’-terminal nucleoside aldehyde in DNA cut with the drug in vitro exceeds 80% of the total 5’ ends. Of the 32P representing nucleoside aldehyde in DNA from NCS-exposed cells, 77% is in TMP; the rest is in AMP >> CMP > GMP, a distribution in excellent agreement with that obtained for in vitro drug-treated DNA. DNA sequencing experiments, using the 340 base pair alphoid DNA fragment isolated from drug-treated cells, show that the pattern of breakage produced by NCS within a defined sequence of DNA in intact cells is similar to that in the in vitro reaction, with a preferential attack at thymidylate residues, but a much higher concentration of the drug was required to cause comparable breakage in intact cells. Furthermore, in cells depleted of glutathione, DNA strand breakage was greatly reduced, confirming thiol involvement in the activation of the drug in cells, as in the in vitro reaction.