Mitochondrial DNA replication was examined in mutants for seven different Saccharomyces cerevisiae genes which are essential for nuclear DNA replication. In cdc8 and cdc21, mutants defective in continued replication during the S phase of the cell cycle, mitochondrial DNA replication ceases at the nonpermissive temperature. Replication is temperature sensitive even when these mutants are arrested in the G1 phase of the cell cycle with alpha factor, a condition where mitochondrial DNA replication continues for the equivalent of several generations at the permissive temperature. Therefore the cessation of replication results from a defect in mitochondrial replication per se, rather than from an indirect consequence of cells being blocked in a phase of the cell cycle where mitochondrial DNA is not normally synthesized. Since the temperature-sensitive mutations are recessive, the products of genes cdc8 and cdc21 must be required for both nuclear and mitochondrial DNA replication. In contrast to cdc8 and cdc21, mitochondrial DNA replication continues for a long time at the nonpermissive temperature in five other cell division cycle mutants in which nuclear DNA synthesis ceases within one cell cycle: cdc4, cdc7, and cdc28, which are defective in the initiation of nuclear DNA synthesis, and cdc14 and cdc23, which are defective in nuclear division. The products of these genes, therefore, are apparently not required for the initiation of mitochondrial DNA replication.