The polymerase subunit (alpha) of Escherichia coli DNA polymerase III holoenzyme and the 3'----5' exonuclease subunit (epsilon) are each less active separately than together in the holoenzyme core (an assembly of alpha, epsilon, and theta subunits). In a complex formed from purified alpha and epsilon subunits, polymerase activity increased 2-fold, and that of the 3'----5' exonuclease increased 10- to 80-fold. The alpha-epsilon complex contains one each of the subunits as does the core. Stimulation of 3'----5' exonuclease activity is due mainly to a greatly increased affinity of the epsilon subunit for the 3'-hydroxyl terminus, resulting from DNA binding by the alpha subunit. Proofreading in the course of DNA synthesis by the alpha-epsilon complex was indistinguishable from that of the core. These findings identify the participation of the alpha subunit in proofreading by polymerase III holoenzyme and suggest that the fidelity of DNA replication may be influenced by the relative levels of the alpha and epsilon subunits in the cell.