DNA polymerase A (I or major) and its stimulative factor were purified from 15-20 kg wet weight of baker's yeast by several procedures, which were varied in order to examine the possible occurrence of proteolysis. The extraction was carried out in the presence of 10 or 3 mM phenylmethylsulfonyl fluoride (PMSF), followed by either batchwise adsorption-elution or column chromatography on DEAE-Sepharose (rapid or time-consuming, respectively). These early steps were followed by column chromatographies on DEAE-, CM-, and heparin-Sepharoses, phosphocellulose, and Sephacryl S-300. Preparations of the polymerase obtained by all the procedures described above showed a single protein band at Mr of about 145,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), unless they had been treated with 2-mercaptoethanol (ME). After ME treatment, however, they showed two protein bands at Mr of about 145,000 and 75,000 in SDS-PAGE, except for those obtained by the procedure involving 10 mM PMSF and the batchwise adsorption-elution. All the preparations described above showed practically the same specific activity. This indicates that in intact cells, the polymerase consisted of a single peptide with Mr of about 145,000, and that after cell disruption, it was artificially hydrolyzed in a limited fashion into two peptides with Mr of about 75,000, which were still active and were linked to each other through a disulfide bond. Preparations of the factor obtained by all the procedures described above showed a single protein band at Mr of about 20,000 in SDS-PAGE before and after ME treatment. The relative activities of the purified polymerase were (100%), 123, 21, 37, 196, and 38% with native and denatured salmon sperm DNA, native and denatured calf thymus DNA, poly(dA-dT), and poly(dA).oligo(dT)10, respectively. With the addition of the purified factor, they were 173, 272, 173, 217, 173, and 247%, respectively, i.e., significantly stimulated. The purified factor also stimulated the activity of calf thymus DNA polymerase alpha by 150% with denatured salmon sperm DNA; Km was about 5 X 10(-10)M, practically the same as that of yeast DNA polymerase A. However, it hardly influenced the activities of Escherichia coli enzyme I or Micrococcus luteus enzyme.