Camptothecin is a potent antineoplastic agent that interferes with the action of eukaryotic DNA topoisomerase I; the covalent enzyme-DNA intermediate is reversibly stabilized, leading to G2 arrest and cell death. We used a genetic screen to identify cellular factors, other than DNA topoisomerase I, that participate in the process of camptothecin-induced cell death. Following ethyl methanesulfonate mutagenesis of top1 delta yeast cells expressing plasmid-borne wild-type DNA topoisomerase I, six dominant suppressors of camptothecin toxicity were isolated that define a single genetic locus, sct1. Mutant SCT1 cells expressed DNA topoisomerase I protein of similar specific activity and camptothecin sensitivity to that of congenic, drug-sensitive sct1 cells, yet were resistant to camptothecin-mediated lethality. Moreover, camptothecin-treated SCT1 cells did not exhibit the G2-arrested, terminal phenotype characteristic of drug-treated wild-type cells. SCT1 cell sensitivity to other DNA-damaging agents suggests that alterations in SCT1 function suppress camptothecin-induced DNA damage produced in the presence of yeast DNA topoisomerase I.