A specific fraction of chromosomal DNA from both yeast and a wide variety of other eukaryotes, but not from Escherichia coli, promotes high-frequency transformation in yeast. The plasmids containing these sequences are maintained as extra-chromosomal molecules in transformed cells. These results suggest that similar or identical sequences are used for the initiation of DNA replication in eukaryotes. To test this hypothesis, several foreign eukaryotic DNAs implicated directly or indirectly in the initiation of DNA replication have been examined for their ability to promote autonomous, extrachromosomal replication in yeast. Simian virus 40 DNA, amplified Xenopus laevis ribosomal DNA, X. laevis 5S ribosomal DNA, X. laevis mtDNA, and five different members of the Alu I family of human middle repetitive DNAs were cloned into the vector YIp5 and used to transform yeast. Of these DNAs, only Xenopus mtDNA promoted high-frequency transformation and extrachromosomal maintenance of YIp5 DNA. A 2.2-kilobase EcoRI fragment from the 17.4-kilobase mtDNA molecule was responsible for these activities. This fragment contains the sequence used for the initiation of replication in Xenopus mitochondria.