We have determined the fidelity of in vitro DNA synthesis catalyzed at high temperature by the DNA polymerase from the thermophilic bacterium Thermus aquaticus. Using a DNA substrate that contains a 3'-OH terminal mismatch, we demonstrate that the purified polymerase lacks detectable exonucleolytic proofreading activity. The fidelity of the Taq polymerase was measured by two assays which score errors produced during in vitro DNA synthesis of the lacZ alpha complementation gene in M13mp2 DNA. In both assays, the Taq polymerase produces single-base substitution errors at a rate of 1 for each 9000 nucleotides polymerized. Frameshift errors are also produced, at a frequency of 1/41,000. These results are discussed in relation to the effects of high temperature on fidelity and the use of the Taq DNA polymerase as a reagent for the in vitro amplification of DNA by the polymerase chain reaction.