There is a DNA sequence which has unusually high affinity for the DnaA protein of Escherichia coli between the glyV and amiB–mutL operons at 94.7 min on the genetic map. Affinity of DnaA protein for DNA was measured in vivo as the activity of β-galactosidase produced from the lacZ gene on a plasmid fused to the 5′-terminal portion of the mioC gene, which is under the control of the DnaA protein. The chromosomal DNA segment between the two operons, carried on a compatible plasmid, derepressed the β-galactosidase activity by titrating DnaA protein. Derepression occurred on the chromosomal dnaA gene as well, since it is autoregulated. Hence, as measured by immunoassays, one plasmid molecule carrying the DnaA-binding region titrated 370 DnaA molecules, which is a value eightfold higher than that for a plasmid containing the oriC region. We estimate that about 60% of the total cellular DnaA molecules are bound to this site. Four DnaA-binding sequences (DnaA boxes) and a DnaA-regulated promoter directing transcription of two small genes were present within a 250 bp stretch in this region but additional long DNA regions, including the fifth DnaA box located about 650 bp downstream, were required for maximum binding. A role for the DnaA-binding site in controlling DnaA-protein concentration in the cell cycle is discussed.