The activity of DnaA protein, the initiator of chromosome replication in Escherichia coli, is regulated by adenine nucleotide binding; the ATP-bound form, not the ADP-bound form, is active. DnaAcos is a mutant protein that is insensitive to negative regulation by ADP. Initiation of chromosome replication occurs excessively in the dnaAcos mutant at 30°C, a restrictive temperature for growth. To determine the control factors that act independently of adenine nucleotide binding of DnaA, we analysed suppressors from the dnaAcos mutant isolated by Tn5 insertion mutagenesis. Three of the suppressors carried Tn5 in the aroK or aroB gene, the first two cistrons in the dam operon. Complementation tests revealed that the dam gene is responsible for the suppression. Over-replication of the chromosome was inhibited in the dnaAcos aroK::Tn5 double mutant, and initiation of chromosome replication in the dnaA+aroK::Tn5 mutant was partially inhibited. The aroK (or B )::Tn5 cells contained DnaA molecules at a level similar to that in the parental aroBK + strain. Moreover, dnaAcos suppression depended on the function of the seqA gene. Thus, Dam activity positively regulates initiation of chromosome replication in vivo. SeqA function seems to be distinguished from the control of DnaA protein by adenine nucleotide binding.