The accuracy of DNA synthesis by DNA polymerase B1 from the hyperthermophilic archaeon Sulfolobus solfataricus (Sso pol B1) at near the physiological temperature was investigated using M13-based mutational assays. Sso pol B1 showed replication fidelity similar to or higher than most viral, bacterial, and eukaryotic replicases. The fidelity of the enzyme was about three times as high at 70 degrees C as at 55 degrees C. Approximately two-thirds of the errors made by the enzyme were single-base substitutions, of which 58% were C --> T transition. Frameshift mutations, mostly resulting from single-base deletions, accounted for 19% of the total errors. An exonuclease-deficient mutant of Sso pol B1 was three times as mutagenic as the wild-type enzyme, suggesting that the intrinsic proofreading function contributed only modestly to the fidelity of the enzyme. Kinetic assays showed that the frequencies of all possible misincorporations by an exonuclease-deficient triple-point mutant of Sso pol B1 ranged from 5.4 x 10(-5) to 4.6 x 10(-4). The high fidelity of this enzyme in DNA synthesis was based primarily on K (m) difference rather than V (max) difference. These properties of Sso pol B1 are consistent with the proposed role of the enzyme as a replicase in S. solfataricus.