The dnaX gene (previously called dnaZX) of Escherichia coli has only one open reading frame for a 71-kDa polypeptide from which two distinct DNA polymerase III holoenzyme subunits, tau (71 kDa) and gamma (47 kDa), are produced. To determine how the gamma subunit is generated, we examined the influence of mutations in the dnaX gene on the pattern of tau and gamma production in overproducing cells. Important structural elements in dnaX mRNA include a stretch of six adenines (nucleotides 1425-1430), a stable hairpin structure (nucleotides 1437-1466), and a UGA stop codon in a -1 frame (nucleotides 1434-1436) between the stretch of adenines and the hairpin structure. Disruption of this stop codon generates a slightly larger gamma subunit, indicative of the use of a -1 stop codon farther downstream (nucleotides 1470-1472). These results suggest that a -1 frameshift during translation allows the use of this UGA codon to terminate translation of the gamma polypeptide. The amino acid composition, sequence, and mass spectra of a C-terminal peptide from mild digestion of the purified gamma protein with endoproteinase Lys-C confirms that this frameshift occurs at either of the two lysine codons in the region of the adenine stretch. Remarkable features of this frameshifting are its high frequency (i.e., about 80% in an overproducing cell) and the striking structural similarity to the frameshifting signal responsible for expression of the pol and pro genes in many retroviruses.