To define the role of the early palindrome domain within the core origin of simian virus 40 (SV40) DNA replication, 800 bp DNA fragment containing SV40 origin of replication was inserted into filamentous phage M13 DNA. Four kinds of mutants were introduced into this recombinant phage DNA, M13 SV-2, by oligonucleotide-directed site-specific mutagenesis. Bacteriophage M13 DNAs carrying the wild-type or base substituted SV40 origin of replication were used for replication assay. In vivo and in vitro assay with african green monkey kidney cell lines showed that the replication of M13-SV40 recombinant DNAs was restricted like a pBR322-SV40 recombinant DNA (Lusky and Botchan, 1981). Furthermore, recombinant phage DNAs isolated from the transfected simian cells subsequently showed a reduced ability to retransform E. coli. Then, the origin fragments containing one or two base substitutions were subcloned into plasmid pAT153, a derivative of pBR322. In two mutants (pATSV-A, -B) the stems were destroyed by single base substitutions. In the other two mutants (pATSV-C, -D) the normal stems were restored by additional base initially. While pATSV-A and pATSV-C could tolerate base substitutions, pATSV-B and pATSV-D showed dramatic decrease of DNA replication in monkey cells. These results suggest that the early palindrome domain does not function as a cruci-form structure, but may be involved in DNA replication initiation as a sequence-specific manner.