Aphidicolin, a known inhibitor of DNA polymerase alpha, is a potent inhibitor of nuclear DNA synthesis in HeLa cells but has no effect on the replication of mitochondrial DNA. Parallel experiments with mitochondria incubated in vitro also show no inhibition of DNA synthesis by aphidicolin; however, DNA synthesis in these isolated mitochondria is completely blocked by dideoxycytidine triphosphate, which inhibits DNA polymerase gamma but not the alpha polymerase. The replication of mitochondrial DNA therefore requires only one DNA polymerase of the gamma type.