Two forms of Escherichia coli DNA polymerase III, DNA polymerase III', and DNA polymerase III have been shown to synthesize DNA products via a processive mechanism with product sizes distinctive for each enzyme form. These forms of DNA polymerase III are intermediate in complexity between the core DNA polymerase III and the DNA polymerase III holoenzyme. In a previous publication (Fay, P. J., Johanson K. O., McHenry, C. S., and Bambara, R. A. (1981) J. Biol. Chem. 256, 976-983), we demonstrated that on a randomly primed fd DNA template or on an oligo(dT)10 . poly(dA) template, the DNA polymerase III holoenzyme adds more than 100 nucleotides before dissociation, whereas the core enzyme adds 10 to 15 nucleotides. Now we show that DNA polymerase III' adds 30 to 40 nucleotides before dissociation. This number can be increased to approximately 60 if spermidine is present, but it is insensitive to the presence of E. coli single-stranded DNA-binding protein. DNA polymerase III adds about 50 nucleotides before dissociation, but this value can be increased to 200 nucleotides in the presence of the binding protein. Using measurement of product sizes made on an oligo(dT)10 . poly(dA) template, reconstitution of holoenzyme activity from DNA polymerase III and the beta subunit was monitored. Finally, it is shown that the products obtained from a purified initiation complex of holoenzyme and oligo(dT)10 . poly(dA) derive solely from the holoenzyme.