ABSTRACT Bacillus subtilis undergoes a highly distinctive division during spore formation. It yields two unequal cells, the mother cell and the prespore, and septum formation is completed before the origin-distal 70% of the chromosome has entered the smaller prespore. The mother cell subsequently engulfs the prespore. Two different probes were used to study the behavior of the terminus ( ter ) region of the chromosome during spore formation. Only one ter region was observed at the time of sporulation division. A second ter region, indicative of chromosome separation, was not distinguishable until engulfment was nearing completion, when one was in the mother cell and the other in the prespore. Separation of the two ter regions depended on the DNA translocase SpoIIIE. It is concluded that SpoIIIE is required during spore formation for chromosome separation as well as for translocation; SpoIIIE is not required for separation during vegetative growth.