The alternative forms of the DnaX protein found in Escherichia coli DNA polymerase III holoenzyme, tau and gamma, were purified from extracts of strains carrying overexpressing plasmids mutated in their frameshifting sequences such that they produced only one subunit or the other. The purified subunits were used to reconstitute the tau and gamma complexes which were characterized by functional assays. The gamma complex-reconstituted holoenzyme required a stoichiometric excess of DNA polymerase III core, beyond physiological levels, for activity. The tau subunit stimulated the gamma complex 2-fold, but could not be used to reconstitute a holoenzyme with gamma complex and stoichiometric quantities of core. In the presence of adenosine 5'-O-(3'-thiotriphospate) (ATP gamma S), the DNA polymerase III holoenzyme behaves as an asymmetric dimer; it can form only initiation complexes with primed DNA in one-half of the enzyme (Johanson, K. O., and McHenry, C. S. (1984) J. Biol. Chem. 259, 4589-4595). An asymmetric distribution of two products of the dnaX gene, gamma and tau, has been postulated to underlie the asymmetry of holoenzyme. To provide a direct test for this hypothesis, we reconstituted holoenzyme containing only the gamma or tau DnaX proteins. We observed that, although gamma could function in the presence of ATP and high concentrations of DNA polymerase III core, it was nearly inert in the presence of ATP gamma S. In contrast, tau-containing holoenzyme behaved exactly like native holoenzyme in the presence of ATP gamma S. These results implicate tau as a key component required to reconstitute holoenzyme with native behavior and show that tau plays a key role in initiation complex formation. These results also show that gamma is not a necessary component, since all of the known properties of native holoenzyme can be reproduced with a 9-subunit tau-holoenzyme.