A human cDNA encoding a previously unrecognized DNA ligase IV has been identified (Wei, Y.-F., Robins, P., Carter, K., Caldecott, K., Pappin, D. J. C., Yu, G.-L., Wang, R.-P., Shell, B. K., Nash, R. A., Schär, P., Barnes, D. E., Haseltine, W. A., and Lindahl, T. (1995) Mol. Cell. Biol. 15, 3206-3216). Antibodies have been raised against predicted peptide sequences of DNA ligase IV and used to identify the enzyme during purification from HeLa cell nuclei. The 96-kDa DNA ligase IV and the 103-kDa DNA ligase III co-migrate during SDS-polyacrylamide gel electrophoresis and have similar column fractionation properties, which complicates the distinction between the two enzymes, but they have been separated by Mono S liquid chromatography. During initial size fractionation by gel chromatography in 1 M NaCl, DNA ligase IV elutes in the same position as the DNA ligase III-XRCC1 protein complex, indicating that DNA ligase IV is also bound to another protein or occurs as a dimer. DNA ligase IV has been purified free from other DNA ligases, and its enzymatic properties have been examined. The purified protein effectively joins single-strand breaks in a double-stranded polydeoxynucleotide in an ATP-dependent reaction. The substrate specificity of DNA ligase IV differs from those of the other two cloned human DNA ligases, I and III, with regard to their ability to join the hybrid substrates oligo(dT).poly(rA) and oligo(rA).poly(dT). DNA ligase IV occurs in part as an enzyme-adenylate complex in HeLa cell nuclear extracts.