The Escherichia coli DnaA protein is a sequence-specific DNA binding protein that promotes the initiation of replication of the bacterial chromosome, and of several plasmids including pSC101. Twenty-eight novel missense mutations of the E. coli dnaA gene were isolated by selecting for their inability to replicate a derivative of pSC101 when contained in a lambda vector. Characterization of these as well as seven novel nonsense mutations and one in-frame deletion mutation are described here. Results suggest that E. coli DnaA protein contains four functional domains. Mutations that affect residues in the P-loop or Walker A motif thought to be involved in ATP binding identify one domain. The second domain maps to a region near the C terminus and is involved in DNA binding. The function of the third domain that maps near the N terminus is unknown but may be involved in the ability of DnaA protein to oligomerize. Two alleles encoding different truncated gene products retained the ability to promote replication from the pSC101 origin but not oriC, identifying a fourth domain dispensable for replication of pSC101 but essential for replication from the bacterial chromosomal origin, oriC.