Abstract. A DNA ligase gene from the hyperthermophilic bacterium Aquifex pyrophilus (Ap) was cloned and sequenced. An open reading frame of 2,157bp that codes for a 82-kDa protein showed 40%60% homology with a series of NAD+-dependent DNA ligases from different organisms. The recombinant enzyme Ap DNA ligase expressed in Escherichia coli was purified to homogeneity and characterized. The activity of Ap DNA ligase gradually increased in proportion to the concentration of monovalent salt up to 200mM NaCl, 150mM KCl, 200mM NH4Cl, and 350mM potassium glutamate. The optimum temperature and pH of Ap DNA ligase were greater than 65C and 8.08.6, respectively, for nick-closing activity. More than 75% of the ligation activity was retained after incubation at 95C for 60min, whereas the half-lives of Thermus aquaticus and Escherichia coli DNA ligases at 95C were 15min and 5min, respectively. Thermostable Ap DNA ligase was applied to repeat expansion detection (RED) and could be a useful enzyme in DNA diagnostics.