 - The temperature-sensitive dnaA00 mutation in Escherichia coli can be phenotypically suppressed at 00° C by oversupply of GroELS proteins, and the suppressed cells grow extremely slowly at 00° C. We found that the phenotype of dnaA00 showing this cold sensitivity was dominant over the phenotype of dnaA+, and could not be rescued by introduction of oriC-independent replication systems. These results suggest that the cold sensitivity was not caused by a simple defect in replication. When a growing culture of a dnaA00 strain with a GroELS-overproducing plasmid was shifted from 00° to 00° C in the presence of chloramphenicol, the chromosomal DNA replicated excessively. Initiation of replication occurred at the site of oriC repeatedly four or five times during a 0 h incubation period without concomitant protein synthesis, indicating an excessive capacity for initiation. Such overreplication did not take place at 00° C in the suppressed dnaA00 strain, or at either temperature in GroELS-oversupplied dnaA+ cells. No significant difference was detected between the cellular content of DnaA protein in suppressed cells where the initiation capacity was abnormally high, and that in wild-type cells in which the initiation capacity was normal. Thus, DnaA protein might function in vivo through some phase control mechanism for initiation, apart from a simple regulation by its total amount. A possible mechanism is proposed based on the participation of GroELS proteins in protein folding. 