A DNA-polymerase from the extreme thermophilic bacteria Thermus flavus has been isolated. The five-step purification procedure resulted in an electrophoretically homogeneous enzyme with molecular weight of 66000. The isolated enzyme is thermostable and a temperature optimum on the DNA templates at 70 degrees and that on RNA templates at 50 degrees. The enzyme does not contain contaminant endo- and exonuclease activities. The maximal activity of the enzyme requires the presence of template, four deoxyribonucleoside triphosphates and monovalent and bivalent cations in the incubation mixture. The enzyme is highly active when "activated" DNA, poly(dA)-poly(dT), poly(dA)-oligo(dT) 10 and poly(rA)-oligo(dT)10 are used as templates, moderately active on single-stranded and double-stranded DNAs and inactive on poly(rC)-oligo(dG)12-18 and native RNA molecules.