The incorporation of thymidine into the DNA of eukaryotic cells is markedly depressed, but not completely inhibited, by aphidicolin, a highly specific inhibitor of DNA polymerase alpha. An electron microscope autoradiographic analysis of the synthesis of nuclear and mitochondrial DNA in vivo in Concanavalin A stimulated rabbit spleen lymphocytes and in Hamster cell cultures, in the absence and in the presence of aphidicolin, revealed that aphidicolin inhibits the nuclear but not the mitochondrial DNA replication. We therefore conclude that DNA polymerase alpha performs the synchronous bidirectional replication of nuclear DNA and that DNA polymerase gamma, the only DNA polymerase present in the mitochondria, performs the "strand displacement" DNA synthesis of these organelles.