Polydeoxyribonucleotide kinase (PNK) is a mammalian DNA repair enzyme that has the capacity to phosphorylate 5' DNA termini and dephosphorylate 3' DNA termini. A series of murine monoclonal antibodies (MAbs) was raised against the full-length recombinant human PNK. Seven of these antibodies were selected and characterized by enzyme immunoassay, Western blot analysis, and their capacity to immunoprecipitate PNK. The epitope location was defined by cyanogen bromide digestion and by using a truncated PNK for Western blot analysis. All of the MAbs recognize a single 60-kDa protein in human cell extracts. PNKs from calf, monkey, and Chinese hamster cell and tissue extracts were also detected by some or all of the MAbs. These antibodies can be successfully used for the cellular, biochemical, and functional analysis of PNK in different mammalian cell lines.