To better understand how the E2F1 transcription factor contributes to the process of cell proliferation, NIH-3T3 cell lines were generated that constitutively express either the wild-type E2F1 protein or an amino terminal deletion mutant, termed E2Fld87. Proliferating E2Fld87-ex-pressing cells exhibit a significant lengthening of S phase relative to control and E2F1 cell lines and are hypersensitive to the cytotoxic effects of the S phase-specific antitumor drug camptothecin. This sensitivity is associated with an increase in drug-induced p53 and WAF1 levels. The E2F1 and E2Fld87 cell lines are both able to initiate, but not complete, S phase under conditions of serum starvation. However, quantitation of DNA synthesis, during culture in serum-deprived media, indicates that the E2Fld87 cell line synthesizes more DNA/cell as compared to the E2F1 cell line. Consistent with this relative increase in DNA synthesis, the E2Fld87 cell line undergoes camptothecin-induced apoptosis when cultured under conditions of serum starvation, while the control and E2F1 cell lines are unaffected by drug treatment under the same conditions. Thus, the sensitivity of the E2Fld87 cell line to camptothecin is not dependent on cell proliferation. The data presented here suggest that cell cycle parameters can be manipulated in order to enhance sensitivity of a cell to the toxic effects of specific chemotherapeutic agents. © 1995, American Association for Cancer Research. All rights reserved.