A polynucleotide kinase, which catalyzes the phosphorylation of 5'-hydroxyl ends of deoxyribonucleic acid in the presence of adenosine triphosphate, was extracted from rat liver nuclei. Its optimal pH was 5.5. It was thermolabile and completely dependent on the presence of Mg2+ for activity. p-Chloromercuribenzoate inactivated this enzyme and this inactivation was overcome by mercaptoethanol. The 32P labeled DNA products of this reaction resulted in the release of 32P by the action of alkaline phosphatase [EC 3.1. 3.1] or deoxyribonuclease [EC 3.1. 4.5]. There is no striking difference in specific activity of this enzyme between normal and partially hepatectomized rat livers.