The minimal region required for expression of the dnaE gene of Escherichia coli has been determined relative to a detailed restriction endonuclease map. This has been accomplished by analysis of Bal 31 exonuclease-generated deletions from the termini of the E. coli DNA contained in plasmid pMWE303 , a plasmid that we have previously demonstrated to contain the dnaE gene (M. M. Welch and C. S. McHenry , J. Bacteriol . 152:351-356, 1982). The competence of these deletion-containing plasmids in expressing the alpha subunit of DNA polymerase III holoenzyme has been determined by their ability both to complement a dnaE mutant and to direct the synthesis of a complete alpha subunit. The carboxyl-terminal coding region of dnaE has been identified through the detection of partial alpha polypeptides encoded by plasmids containing deletions from one end of the gene. This approach has permitted the precise determination of both termini of the dnaE gene and the determination of the orientation of the gene within the E. coli chromosome.