Mutants of the yeast, Saccharomyces cerevisiae, deficient in DNA topoisomerase I activity have been identified. One mutant has normal topoisomerase I activity when assayed at 25 degrees C and about 20% of normal activity when assayed at 36 degrees C. Strains with this mutation grow normally at all temperatures tested. The mutation has been mapped to MAK1, a gene required for maintenance of killer RNA. Three previously isolated mak1 mutants exhibit less than 1% of normal topoisomerase I activity in our assay, but yet they grow normally. The implications of these results for the role of DNA topoisomerase I in the cell are discussed.