A rapid, three-step purification of DNA alpha-polymerase from calf thymus is described. The key feature is immunoaffinity chromatography using a column of immobilized monoclonal immunoglobulin G (IgG) developed against human KB cell alpha-polymerase. This step is followed by preparative sucrose gradient sedimentation. The highly purified polymerase has a specific activity of 35 000 nmol of nucleotide incorporated per hour per milligram. Its molecular weight is 404 000. This molecular weight is higher than observed in some earlier purifications, possibly because salt concentrations are kept at nearly physiological levels. Also, the rapidity of purification in the presence of multiple protease inhibitors minimizes degradation. The purified enzyme is inhibited by aphidicolin, N-ethylmaleimide, and the specific monoclonal IgG, thereby identifying it as DNA alpha-polymerase. ATP at 4 mM concentration stimulates enzymatic activity up to 4-fold on calf thymus DNA templates. The enzyme is also capable of priming single-stranded DNA with RNA. The procedure represents a significant advance from purifying alpha-polymerase from calf by conventional means, since it avoids ion-exchange chromatography and harsh conditions. It also minimizes the time required to produce sufficient quantities of purified high molecular weight polymerase for analysis.