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A temperature-sensitive mutant of Bacillus subtilis, CRK2001, defective in the initiation of DNA replication is described. When the temperature was raised, initiation of DNA replication was immediately prevented, while on-going replication continued to complete rounds of replication. Reinitiation occurred from the normal replication origin 10 to 15 minutes after cells heated to 47°C were cooled to 30°C. The reinitiation did not require protein synthesis but did require de novo RNA synthesis. The ability of the cell to reinitiate replication either at 47°C or in the presence of rifampicin returned very rapidly at 30°C. Using a rifampicin-reversible derivative of CRK2001 it could be shown that a temperature-resistant initiation potential (cell's ability for initiating replication at 47°C) was formed at 30°C just as rapidly in the presence of rifampicin as in its absence. This indicates that both reactivation of a temperature-sensitive product (Pten7) and de novo synthesis of initiation RNA are required for the reinitiation. I-RNA ‡ synthesis occurred at 47°C as well as at 30°C, but it turns over rapidly with a half-life of two minutes in the absence of the active gene product. The half-life of I-RNA in this state was appoximately the same as that of average messenger RNA. A completed initiation potential was formed at 30°C in the absence of thymine. It remained stable at 47°C but decayed slowly in the presence of rifampicin. The half-life of RNA in this stage was 12 minutes and was much longer than that of I-RNA synthesized in the absence of active gene product. A sequence of events involved in the formation of the initiation potential in this mutant was constructed based on these observations.