The efficient repair of double-strand breaks (DSBs) in DNA is critical for the maintenance of genome stability. In mammalian cells, repair can occur by homologous recombination or by non-homologous end joining (NHEJ). DNA breaks caused by reactive oxygen or ionizing radiation often contain non- conventional end groups that must be processed to restore the ligatable 3'-OH and 5'-phosphate moieties which are necessary for efficient repair by NHEJ. Here, using cell-free extracts that efficiently catalyse NHEJ in vitro, we show that human polynucleotide kinase (PNK) promotes phosphate replacement at damaged termini, but only within the context of the NHEJ apparatus. Phosphorylation of terminal 5'-OH groups by PNK was blocked by depletion of the NHEJ factor XRCC4, or by an inactivating mutation in DNA-PK(cs), indicating that the DNA kinase activity in the extract is coupled with active NHEJ processes. Moreover, we find that end-joining activity can be restored to PNK-depleted extracts by addition of human PNK, but not bacteriophage T4 PNK. This work provides the first demonstration of a direct, specific role for human PNK in DSB repair.