The cell-free synthesis of DNA polymerase in translation mixtures containing calf thymus total and poly (A+) RNA was examined using activity gel analysis and immunobinding with a monoclonal antibody to calf thymus alpha-polymerase. Activity gel analysis indicated that functional DNA polymerase catalytic polypeptides of Mr = 110,000 to 120,000 and Mr approximately 68,000 were synthesized. Sucrose density gradient centrifugation of total RNA resulted in resolution and partial purification of the mRNAs encoding these two DNA polymerase polypeptides. Immunobinding experiments with the monoclonal antibody to calf alpha-polymerase confirmed that an immunoreactive polypeptide of 110 to 120 kilo-daltons had been formed in vitro. This polypeptide and the 68,000-Mr polypeptide correspond in size to alpha-polymerase catalytic polypeptides observed in crude extracts of calf cells and in purified calf alpha-polymerase.