The replication origin of the E. coli K-12 chromosome has been isolated as autonomously replicating molecules(oriC plasmid), and the DNA region essential for replicating function(oriC) has been localized to a sequence of 232-245 base-pairs(bp) by deletion analysis. In this report, the functional role of oriC was analysed by using an in vitro replication system and various OriC+ and OriC- plasmids previously constructed. The results obtained were summarized as follows: (1) The oriC sequence contained information enough to direct bidirectional replication. (2) The actual DNA replication began at a region near, but outside, oriC and progressed bidirectionally. (3) Initiation of DNA synthesis at the specific region required the dnaA-complementing fraction from cells harboring a dnaA-carrying plasmid.