The function of the theta subunit of Escherichia coli DNA polymerase III holoenzyme is not well established. theta is a tightly bound component of the DNA polymerase III core, which contains the alpha subunit (polymerase), the epsilon subunit (3'-->5' exonuclease), and the theta subunit, in the linear order alpha-epsilon-theta. Previous studies have shown that the theta subunit is not essential, as strains carrying a deletion of the holE gene (which encodes theta) proved fully viable. No significant phenotypic effects of the holE deletion could be detected, as the strain displayed normal cell health, morphology, and mutation rates. On the other hand, in vitro experiments have indicated the efficiency of the 3'-exonuclease activity of epsilon to be modestly enhanced by the presence of theta. Here, we report a series of genetic experiments that suggest that theta has a stabilizing role for the epsilon proofreading subunit. The observations include (i) defined DeltaholE mutator effects in mismatch-repair-defective mutL backgrounds, (ii) strong DeltaholE mutator effects in certain proofreading-impaired dnaQ strains, and (iii) yeast two- and three-hybrid experiments demonstrating enhancement of alpha-epsilon interactions by the presence of theta. theta appears conserved among gram-negative organisms which have an exonuclease subunit that exists as a separate protein (i.e., not part of the polymerase polypeptide), and the presence of theta might be uniquely beneficial in those instances where the proofreading 3'-exonuclease is not part of the polymerase polypeptide.