Mouse liver DNA was cut out with BamHI and cloned into YIp5, which contained the URA3 gene of Saccharomyces cerevisiae in pBR322. Of the several plasmids isolated, two plasmids, pMU65 and pMU1l1, could transform S. cerevisiae from the URA-to the URA+ phenotype and could replicate autonomously within the transformant, indicating that mouse DNA fragments present in pMU65 or pMU111 contain autonomously replicating sequences (ARS) for replication in S. cerevisiae. Furthermore, to determine the correlation between ARS function in yeast cells and that in much higher organisms, we tried to challenge these plasmids with the simian virus 40 (SV40) DNA replication system. Of the two plasmids tested, the EcoRI-Bglll region of pMU65 could be hybridized with a chemically synthesized 13-nucleotide fragment corresponding to the origin region of SV40 DNA. Both pMU65 (the EcoRI-BglH region cloned in pBR322) and its subclone pMU65EB could replicate semiconservatively, and initiation of DNA replication started from the EcoRI-BglIH region when the replicating activity of these plasmids was tested in the in vitro SV40 DNA replication system we have established before. Furthermore, pMU65 and pMU65EB could replicate autonomously within monkey Cos cells which produce SV40 T antigen constitutively. These results show that a 2.5-kilobase fragment of the EcoRI-Bglll region in pMU65 contains the ARS needed for replication in the SV40 DNA replication system.The replication of eucaryotic DNA occurs by initiation at many chromosomal sites which are usually clustered in a specific region characteristic of the cell type and the stage of the cell cycle (7,9,12,19,43). Recently, several investigators have tried to isolate replication origins by using in vitro recombination techniques (1,16,21,41). Another approach to isolation of the replication origin is to get autonomous replicating sequences (ARS). Many eucaryotic ARS of yeasts, as well as those of other higher organisms, have been described (8, 10, 14, 18, 20-22, 24, 26, 28, 29, 33, 34, 37-40, 44, 45; J. M. Vallet, M. Rahire, and J. D. Rochaix, EMBO J., in press). These sequences from higher organisms may act as initiators of replication in yeast cells. However, there is no evidence that these cloned eucaryotic DNA segments can also act as replicators in cells of the original higher organism. Another approach to finding the replication origin is to search for the sequences similar to the origin of the eucaryotic virus genome, because the regulatory sequences might be conserved over the species (25). Simian virus 40 (SV40) DNA seems to resemble one replicon of the eucaryotic genome. Therefore, several investigators tried to find, in monkey or human cells, sequences similar to that of the SV40 origin (11,27,30). We have already developed the in vitro system in which initiation of DNA replication occurs on exogenous DNA containing the SV40 origin (4). In this system, the human Alu family, which is one of interspersed repeated sequences (35), showed a good template activity, and the initiation s...