Previously, we localized the beta2 interacting portion of the catalytic subunit (alpha) of DNA polymerase III to the C-terminal half, downstream of the polymerase active site. Since then, two different beta2 binding sites within this region have been proposed. An internal site includes amino acid residues 920-924 (QADMF) and an extreme C-terminal site includes amino acid residues 1154-1159 (QVELEF). To permit determination of their relative contributions, we made mutations in both sites and evaluated the biochemical, genetic, and protein binding properties of the mutant alpha subunits. All purified mutant alpha subunits retained near wild-type polymerase function, which was measured in non-processive gap-filling assays. Mutations in the internal site abolished the ability of mutant alpha subunits to participate in processive synthesis. Replacement of the five-residue internal sequence with AAAKK eliminated detectable binding to beta2. In addition, mutation of residues required for beta2 binding abolished the ability of the resulting polymerase to participate in chromosomal replication in vivo. In contrast, mutations in the C-terminal site exhibited near wild-type phenotypes. alpha Subunits with the C-terminal site completely removed could participate in processive DNA replication, could bind beta2, and, if induced to high level expression, could complement a temperature-sensitive conditional lethal dnaE mutation. C-terminal defects that only partially complemented correlated with a defect in binding to tau, not beta2. A C-terminal deletion only reduced beta2 binding fourfold; tau binding was decreased ca 400-fold. The context in which the beta2 binding site was presented made an enormous difference. Replacement of the internal site with a consensus beta2 binding sequence increased the affinity of the resulting alpha for beta2 over 100-fold, whereas the same modification at the C-terminal site did not significantly increase binding. The implications of multiple interactions between a replicase and its processivity factor, including applications to polymerase cycling and interchange with other polymerases and factors at the replication fork, are discussed.