The yeast Saccharomyces cerevisiae has been useful in establishing the phenotypic effects of specific mutations on the enzymatic activity and camptothecin sensitivity of yeast and human DNA topoisomerase I. To determine whether these phenotypes were faithfully reiterated in higher eukaryotic cells, wild-type and mutant yeast Top1 proteins were epitope-tagged at the amino terminus and transiently overexpressed in mammalian COS cells. Camptothecin preferentially induced apoptosis in cells expressing wild-type eSctop1p yet did not appreciably increase the cytotoxic response of cells expressing a catalytically inactive (eSctop1Y727F) or a catalytically active, camptothecin-resistant eSctop1vac mutant. Using an epitope-specific antibody, immobilized precipitates of eScTop1p were active in DNA relaxation assays, whereas immunoprecipitates of eScTop1Y727Fp were not. Thus, the enzyme retained catalytic activity while tethered to a support. Interestingly, the mutant eSctop1T722A, which mimics camptothecin-induced cytotoxicity in yeast through stabilization of the covalent enzyme-DNA intermediate, induced apoptosis in COS cells in the absence of camptothecin. This correlated with increased DNA cleavage in immunoprecipitates of eScTop1T722Ap, in the absence of the drug. The observation that the phenotypic consequences of expressing wild-type and mutant yeast enzymes were reiterated in mammalian cells suggests that the mechanisms underlying cellular responses to DNA topoisomerase I-mediated DNA damage are conserved between yeast and mammalian cells.