We identified the 1.6-kb region of Thermus thermophilus plasmid pTT8 capable of autonomous replication, which shows a significant sequence similarity to the replicon regions of the ColE2-related plasmids. We showed the requirement of DNA polymerase I for pTT8 replication. The putative rep gene coding for the replication initiator protein, Rep, similar to those of the ColE2-related plasmids was cloned into an expression vector. The 6xHis-Rep protein expressed in Escherichia coli was successfully purified by stepwise denaturing with urea and refolding in the presence of glycerol on Ni-resin. We identified the nucleotide sequence recognized by the pTT8 Rep protein by the SELEX experiment using the purified protein, and proposed the existence of the third origin of pTT8 replication different from those predicted previously. (c) 2006 Elsevier Inc. All rights reserved.