The beta subunit of the DNA polymerase III holoenzyme has been purified 10,000-fold to homogeneity from Escherichia coli HMS-83. The native and denatured molecular weights of beta are 72,000 and 37,000, as determined by equilibrium sedimentation. Thus, beta appears to exist as a dimer when in a state free of other holoenzyme components. This conclusion is supported by the native molecular weight calculated from the sedimentation coefficient (5.0 S) and the Stokes radius (32.5 A) and subunit molecular weights determined from denaturing polyacrylamide gel electrophoresis. An antibody directed specifically against the beta subunit has been prepared and found to block the DNA polymerase III holoenzyme-catalyzed reaction, but not reactions which require only the core DNA polymerase III. Furthermore, this antibody blocks the initiation, but not the elongation reaction catalyzed by the DNA polymerase III holoenzyme. Thus, beta may only be required for formation of the initiation complex between polymerase components and a primed template. Immunoprecipitation studies demonstrate that the beta subunit forms a complex with other holoenzyme components in solution, in the absence of DNA.