Bacterial chromosome replication is tightly regulated at the initiation stage to coordinate with mass increase. Together with chromosome partition at cell division, this regulation mechanism ensures the proper number of chromosomes in daughter cells at any growth rate. Therefore, elucidation of this regulation mechanism is important for understanding the bacterial cell cycle. Despite much effort in Escherichia coli and Bacillus subtilis for many years, the mechanism remains to be completely elucidated. In E. coli, it is proposed that a critical amount of DnaA protein determines the time of initiation of replication in the cell cycle. Our study strongly suggested that this might not be the case in B. subtilis. Recently, remarkable progress has been made in bacterial cytology. The new techniques enable us to examine the subcellular location of proteins of interest and DNA regions of the chromosome (for example, the replication origin) and, therefore, to determine directly when in the cell division cycle and where within the cell initiation of chromosome replication takes place. Using the techniques, we detected the initiation complex by examining subcellular location of several Dna- initiation proteins in B. subtilis. Based on our new findings, we propose a novel model for regulation of the time of initiation of chromosome replication in the cell cycle.