A temperature-sensitive DNA topoisomerase II mutant of the yeast Saccharomyces cerevisiae has been identified. Genetic analysis shows that a single recessive nuclear mutation is responsible for both temperature-sensitive growth and enzymatic activity. Thus, topoisomerase II is essential for viability and the mutation is most probably in the structural gene. Experiments with synchronized mutant cells show that at the nonpermissive temperature cells can undergo one, and only one, round of DNA replication. These cells are arrested at medial nuclear division. Analysis of 2-microns plasmid DNA from these cells shows it to be in the form of multiply intertwined catenated dimers. The results suggest that DNA topoisomerase II is necessary for the segregation of chromosomes at the termination of DNA replication.