Approximately 2,500-fold purifications of DNA polymerase-beta from the nuclear fraction of blastulae of the sea urchin, Hemicentrotus pulcherrimus, was performed. The enzyme preparation, which was devoid of DNase and terminal deoxynucleotidyl transferase as contaminants, showed a sedimentation constant of 3.0 S in a sucrose density gradient, a molecular weight of 50,000 by gel filtration, and an isoelectric point of pH 8.1. The enzyme activity was resistant to sulfhydryl group inhibitors. Its optimal pH was 9.0-9.5 in Tris-maleate buffer and 10.0 in glycine buffer. The optimal NaCl concentration for the activity was 30-60 mM and about half of the activity remained at 0.4 M NaCl. As a template-primer, the enzyme preferred synthetic homopolymers to activated DNA. The order of this preference was as follows; poly (dA)-oligo (dT)12-18 greater than poly (rA)-oligo (dT)12-18 greater than activated DNA. The above results indicate that the enzyme corresponds to DNA polymerase-beta from vertebrate cells.