A novel and non-ubiquitous thermostable DNA polymerase in Thermus antranikianii was expressed in E. coli, isolated and biochemically characterized. The enzyme here referred to as Thermophi, has a C-terminal polymerase domain and a proofreading 3′→5′ exonuclease domain, but lack the 5′→3′ exonuclease domain. The corresponding gene is apparently only found in some but not all Thermus strains. The initial rate of specific activity of this polymerase on nicked DNA was about 360,000 U/mg protein. The optimum activity was found at 55 °C, pH 8.5 and 1.5 mM Mg+2. The polymerase was stable at 70 °C and lost 50% of its activity after 5 min at 85 °C, but could be stabilized above 80 °C by addition of 0.5 M L-proline. A pronounced strand-displacement activity was indicated by the large amount of DNA produced by the enzyme after an overnight, isothermal incubation in presence of hexamer primers. Both single and double stranded DNA was isothermally amplified by the enzyme. The amplified DNA was large and apparently highly branched material and composed of both single and double stranded DNA. The produced material could be partly digested by T7 enonuclease I but it was difficult to cut with common restriction enzymes. Amplification of selected genes from dilute samples was successfully demonstrated with the human β-actin gene. Good amplification was also found with 5 microsatellite markers from salmon DNA. Thermophi amplifies DNA by orders of magnitude but upon extended reaction time the DNA becomes very large and highly branched. It is composed of both single and double strands and then correctly amplified sequences only represent about 10-20% of the total DNA, and long stretches of TATATA repeats frequently occur in the amplified DNA.