Serial studies of human leukemic lymphoblasts (CCRF-CEM line) cultured with 0.25 to 2.5 microM VP-16-213 for 0 to 6 hr indicated that the mechanism of cytotoxicity of this compound involves a primary effect on DNA. The most striking early change shown by flow cytometry in VP-16-213-treated cells was a delay in S-phase transit before arrest of cells in G2. Coinciding with this S-phase delay was a selective inhibition of thymidine incorporation into DNA as well as concentration-dependent scission of DNA strands. Using alkaline elution methods, we were able to detect DNA breakage at concentrations of VP-16-213 well below the level required to demonstrate kinetic effects or inhibition of DNA synthesis. These data suggest that DNA strand scission is the initial event in the sequence of kinetic and biosynthetic changes leading to growth inhibition and death of VP-16-213-treated cells. Inhibition of replicon initiation due to strand scission is a plausible explanation for the cytotoxic action of this podophyllotoxin derivative.