A new form of DNA polymerase III, termed Pol III star (Pol III(*)), has been purified to homogeneity from Escherichia coli. Pol III(*) is temperature sensitive when isolated from a thermo-sensitive dnaE mutant, as had been described for Pol III. Pol III(*) and Pol III are separable by gel filtration. Pol III(*) utilizes a duplex template containing short gaps with the same catalytic properties as Pol III. However, Pol III(*) is able to replicate long, singlestranded templates such as homopolymer chains and viral circles of M13 and varphiX174 if provided with the following: spermidine, a primer fragment, and a new protein, termed copolymerase III(*) (Copol III(*)). The latter, purified to homogeneity, has no known independent enzymatic activity and supports synthesis by Pol III(*) but not by Pol I, Pol II, or Pol III.