We identified a DNA polymerase with properties that differed from those of alpha-like DNA polymerase (Pol I), in Pyrococcus furiosus, a hyperthermophilic archaeon. The novel DNA polymerase (Pol II) was partially purified and characterized. The DNA polymerizing activity of Pol II was relatively sensitive to dideoxythymidine-triphosphate (ddTTP) and it was inhibited by N-ethylmaleimide, but not by aphidicolin. Activity staining gel electrophoresis showed that the DNA polymerizing activity was derived from a polypeptide with a molecular weight of 130000 on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The DNA elongation ability of Pol II using a natural DNA template was much lower than that of Pol I from this organism and DNA synthesis of Pol II seems to be non-processive.