Regions of bacterial chromosomes occupy characteristic locations within the cell. In Bacillus subtilis, the origin of replication, oriC, is located at 0°/360° on the circular chromosome. After duplication, sister 0° regions rapidly move to and then reside near the cell quarters. It has been hypothesized that origin function or oriC sequences contribute to positioning and movement of the 0° region. We found that the position of a given chromosomal region does not depend on initiation of replication from the 0° region. In an oriC mutant strain that replicates from a heterologous origin (oriN) at 257°, the position of both the 0° and 257° regions was similar to that in wild-type cells. Thus, positioning of chromosomal regions appears to be independent of which region is replicated first. Furthermore, we found that neither oriC sequences nor the replication initiator DnaA is required or sufficient for positioning a region near the cell quarters. A sequence within oriC previously proposed to play a critical role in chromosome positioning and partitioning was found to make little, if any, contribution. We propose that uncharacterized sites outside of oriC are involved in moving and/or maintaining the 0° region near the cell quarters.