In this report we describe the purification and structural characterization of a near homogeneous preparation of DNA polymerase-alpha that we have obtained from cultured human KB cells. When analyzed by nondenaturing gel electrophoresis, velocity gradient centrifugation, and isoelectric focusing, the enzyme activity demonstrates a constancy of gel mobility, sedimentation coefficient, and isoelectric point during the final three chromatographic steps of the purification. Native gel electrophoresis of the penultimate polymerase fraction at seven concentrations of acrylamide reveals co-migration of the enzyme activity with a single discernible protein band, at constant specific activity, and indicates that the polymerase protein is electrophoretically homogeneous at this stage. The purified enzyme has a sedimentation coefficient of 7.1 to 7.2 S at high or low ionic strength, a molecular weight gel filtration of 149,000, and an isoelectric point of pH 5.0 to 5.2. Sodium dodecyl sulfate gel analyses demonstrate that the polymerase-alpha molecule is a dimer comprised of two dissimilar subunits of 76,000 and 66,000 daltons that are present in equimolar ratio.