A mitochondrial type I-like, ATP-independent, DNA topoisomerase, isolated from highly purified yeast mitochondria, is genetically related to nuclear topoisomerase I. We found that the mitochondrial topoisomerase activity cannot be detected in yeast mitochondrial extracts prepared from strains in which the topoisomerase I gene (TOP1) is disrupted. Thus, the topoisomerase activity associated with mitochondria is dependent upon the expression of the nuclear topoisomerase I gene.