The DNA polymerase alpha from regenerating rat liver has been purified to near homogeneity. The most highly purified fraction gave a single stained band on native polyacrylamide gel electrophoresis, and DNA polymerase alpha activity was coincident with this band. On pore gradient gel electrophoresis, a molecular radius of 72 A was obtained for the enzyme. On denaturing sodium dodecyl sulfate-polyacrylamide gel, five polypeptides were reproducibly resolved, corresponding to molecular weights of 156,000, 64,000, 61,000, 58,000, and 54,000. The catalytic subunit correlated with the 156,000-dalton polypeptide. In the native state, the separated 54,000- to 64,000-dalton polypeptides interacted among themselves to constitute a hetero oligomer of apparent high molecular weight without DNA polymerase activity. Electron microscopy studies of the enzyme confirmed the biochemical results. The specific activity of the isolated catalytic subunit was in all cases lower than when it was associated with the 54,000- to 64,000-dalton structure.