Thermophilic and thermostable DNA ligase was purified to near homogeneity from the extract of Thermus thermophilus HB8. The purified enzyme has an isoelectric point at pH 6.6 and consists of a single polypeptide of about 79,000 in molecular weight on the bases of sodium dodecyl sulfate-polyacrylamide gel electrophoresis data and an equilibrium sedimentation method. The enzyme requires divalent cations, Mg2+ or Mn2+, and the optimum concentration of these ions being 5-9 X 10(-3) M and 3-6 X 10(-3) M, respectively. The enzyme also requires NAD as a cofactor. The apparent Km for NAD is 1.85 X 10(-8) M and that of (dT)10 is 1.4 X 10(-4) M. The pH optimum is 7.4-7.6 in Tris-HCl and 8.0 in collidine/HCl buffer. The joining reaction is activated by K+ and NH+4 at a concentration of 2-100 mM and inhibited by Na+ above 25 mM. The optimum temperatures of the joining of thymidylate oligomers in the presence of poly(dA) as a template are 27.5 degrees C for p(dT)s, 34.5 degrees C for p(dT)10, and 37 degrees C for p(dT)12-18 and that of cohesive-end DNA restriction fragments is 24-37 degrees C. The nick-closing activity of the enzyme was observed over a wide range of the temperature from 15 to 85 degrees C and the optimum temperature is 65-72 degrees C. The temperature dependency of ligation with HB8 DNA ligase for various substrates was found to shift to a region of 7-10 degrees C higher than that of T4 DNA ligase and the activity of HB8 DNA ligase decreased remarkably below 4 degrees C. The enzyme was stable for 1 week at 37 degrees C, its activity dropped by 50% within 2 days at 65 degrees C.