We have identified a previously reported open reading frame (ORF13) that maps between pepA and valS at 96.6 centisomes of the Escherichia coli genome as the structural gene for the chi subunit of DNA polymerase III holoenzyme. This conclusion is supported by a perfect match of the amino-terminal 24 residues of chi with the DNA sequence of ORF13 and a demonstration that ORF13 directs expression of a protein that co-migrates with authentic chi on SDS-polyacrylamide gels. ORF13, designated holC, was isolated from the E. coli chromosome and inserted into a tac promoter-based expression plasmid to direct production of the chi subunit to 5-7% of the total soluble protein. The 3' end of holC was sequenced to resolve discrepancies between two published versions.