The Escherichia coli DnaA protein, as a sequence-specific DNA binding protein, promotes the initiation of chromosomal replication by binding to four asymmetric 9-mer sequences termed DnaA boxes in oriC. Characterization of N-terminal, C-terminal, and internal in-frame deletion mutants identified residues near the C terminus of DnaA protein required for DNA binding. Furthermore, genetic and biochemical characterization of 11 missense mutations mapping within the C-terminal 89 residues indicated that they were defective in DNA binding. Detailed biochemical characterization of one mutant protein bearing a threonine to methionine substitution at position 435 (T435M) revealed that it retained only nonspecific DNA binding activity, suggesting that threonine 435 imparts specificity in binding. Finally, T435M was inactive on its own for in vitro replication of an oriC plasmid but was able to augment limiting levels of wild type DnaA protein, consistent with the proposal that not all of the DnaA monomers in the initial complex are bound specifically to oriC and that direct interaction occurs among monomers.