Thermostable Tte DNA-polymerase was isolated from the strain Thermus thermophilus B35 which was found in hot spring water. The enzyme with molecular mass 87 kD was isolated using sequential chromatography on DEAE-Sepharose, hydroxylapatite, hexyl-agarose, and heparin-Sepharose. Biochemical properties of Tte DNA-polymerase are similar to those of Tth DNA-polymerase isolated from Thermus thermophilus HB8; however, practical application of Tte-Pol seems to be more favorable due to higher temperature optimum of this enzyme and lack of restriction endonucleases in the initial strain.