The origin of replication of the Escherichia coli chromosome (oriC) is located in an intercistronic region between the gidA and the mioC genes. The possibility that transcription from the promoters of these two genes is involved in minichromosome replication was examined. Inactivation of the gid promoter led to a reduction in transformation frequency with an oriC plasmid but inactivation of the mioC promoter did not. The decrease in transformation frequency was most pronounced when both promoters were inactive. Under conditions that selected for plasmid-harboring cells, mutation of the gid promoter caused efficient multimerization or integration of oriC plasmids into the chromosomal oriC region and loss of free plasmid molecules. These changes in plasmid structure were also observed, albeit less frequently, with some plasmids defective in mioC promoter activity. In an in vitro DNA replication system for oriC DNA, plasmids with a defective gid promoter had greatly reduced template activity and essentially no replication occurred when both promoters were inactive. These results suggest that coupled transcription starting from the gid as well as the mioC promoter activates initiation of plasmid replication, the major contribution being made by gid transcription. These two promoters are suggested to be under stringent control.