The replication origin region of the Escherichia coli K-12 chromosome has been cloned, and a region of 245 base-pairs has been shown to contain all the information for autonomous replication (defined ori). In order to obtain further information on the sequence organization in the defined ori stretch, various types of mutation were introduced by in vitro techniques at a restriction site (AvaII site) which locates near the middle of ori. When the correlation between these mutations and replicating function was examined, different effects were obtained with the types of mutation: the replicating function was completely destroyed by either insertion or deletion of short sequences, but not by base-substitutions. Based on these observations and on the fact that multi-gene products are involved in the initiation of replication, we assume that two categories of sequences are present within the ori stretch, one specifying interaction with initiation factors (recognition sequences) and the other spacing the recognition sequences in appropriate distances (spacer sequences), and that the AvaII site is located in the spacer region.