Pfu, the DNA polymerase from Pyrococcus furiosus, has the lowest error rate of any known polymerase in polymerase chain reaction (PCR) amplification. Previously the protein has been purified from P. furiosus bacterial cultures, and a recombinant form has been produced in a baculovirus system. We have produced a pET plasmid for expression of Pfu in Escherichia coli (the expression plasmid pETpfu is available from ATCC, Accession No. 87496) and found that this plasmid is toxic or unstable in the expressing strain BL21(DE3), even in the absence of induction. However, the plasmid was stable in BL21(DE3) containing the pLysS plasmid, which suppresses expression prior to induction, and a 90-kDa protein was expressed upon addition of isopropyl beta-D-thiogalactopyranoside. The protein was purified by heating (to denature E. coli proteins), followed by chromatography on P11 phosphocellulose and mono Q columns. The purified protein had the same activity as the commercially obtained baculovirus-expressed Pfu in both DNA polymerase and PCR reactions. This bacterial expression system appears to be the method of choice for production of Pfu.