Damage to DNA can result in strand breaks with 58-hydroxyl and 38-phosphate termini. Before DNA polymerases and ligases can rejoin the broken strands, such termini have to be restored to 58-phosphate and 38-hydroxyl groups. Polydeoxynucleotide kinase is an enzyme that may fulfil this function. We have purified the kinases from calf thymus and rat liver to near homogeneity. Based on SDS-polyacrylamide gel electrophoresis and activity gels, the enzymes from both sources are ,60-kDa polypeptides. Both enzymes have an acidic pH optimum (5.5-6.0) for kinase activity, and similar pI values (8.5-8.6), and a specificity for DNA. The calf thymus kinase possesses a 38-phosphatase activity, as has previously been shown for the rat liver enzyme. The minimum size of oligonucleotide that can be labelled is 7-8 nucleotides in length, but the optimal size appears to be .18 nucleotides. Comparison of phosphorylation of oligo(dA) 24 and oligo(dT) 24 with oligonucleotides containing a varied nucleotide sequence indicated that the homopolymers are poorer substrates. Unlike the bacteriophage T4 polynucleotide kinase, the mammalian kinases exhibit no preference for 58-overhanging termini when acting at DNA termini produced by restriction enzymes. With doublestranded oligonucleotide complexes designed to model single-strand gaps and nicks, the mammalian kinases preferentially phosphorylate the 58-terminus associated with the gap or nick, in keeping with the idea that the kinases are involved in the repair of DNA single-strand breaks.