DNA topoisomerases are the molecular targets of a range of anticancer and antimicrobial therapeutics. Many of these drugs act by converting their target enzyme to a DNA-damaging agent through the trapping of the covalent enzyme/DNA intermediate. This drug-mediated trapping of the intermediate is reversible, and the lesion in the DNA disappears on removal of the drug. This reversibility leads to questions concerning how treatment with these drugs effects cell killing. Using drug-permeable yeast strains, we demonstrate that yeast cells arrested in G1 are refractory to drugs that trap the covalent complex between DNA and either topoisomerase I or topoisomerase II. The cell cycle regulation of topoisomerase II is not responsible for the insensitivity of G1 cells to drugs targeting this enzyme because ectopic expression of the enzyme in G1 does not alter drug sensitivity. Commitment to cell killing by anti-topoisomerase II agents predominantly occurs in S phase cells but can also occur as cells progress from G2 through mitosis to G1. We also demonstrate that yeast cells treated with DNA replication inhibitors such as aphidicolin lose sensitivity to camptothecin but not to topoisomerase II-targeting DNA-damaging agents. Our results suggest that DNA synthesis is a major determinant for cell killing by camptothecin but that other S phase-associated processes can effect cytotoxicity by drugs that convert topoisomerase II to a DNA-damaging agent.