In bacteria, DNA replication initiation is tightly regulated in order to coordinate chromosome replication with cell growth. In Escherichia coli, positive factors and negative regulatory mechanisms playing important roles in the strict control of DNA replication initiation have been reported. However, it remains unclear how bacterial cells recognize the right time for replication initiation during the cell cycle. In the Gram-positive bacterium Bacillus subtilis, much less is known about the regulation of replication initiation, specifically, regarding negative control mechanisms which ensure replication initiation only once per cell cycle. Here we report that replication initiation was greatly enhanced in strains that had the origin of replication (oriC) relocated to various loci on the chromosome. When oriC was relocated to new loci further than 250 kb counterclockwise from the native locus, replication initiation became asynchronous and earlier than in the wild-type cells. In two oriC-relocated strains (oriC at argG or pnbA, 25 degrees or 30 degrees on the 36 degrees chromosome map, respectively), DnaA levels were higher than in the wild-type but not enough to cause earlier initiation of replication. Our results suggest that the initiation capacity of replication is accumulated well before the actual time of initiation, and its release may be suppressed by a unique DNA structure formed near the native oriC locus.