In Escherichia coli cells, there is a protein that specifically binds to DNA replication terminus (ter) sites on the host and plasmid genome and then blocks progress of the DNA replication fork. We reported that extract of the cells carrying the plasmid with the tau gene, which was identified to be an essential gene for the termination reaction at the ter site, contained about an 8-fold increase in ter-binding activity of the plasmid-free cells. With improvement of the promoter region of the tau gene on the plasmid by site-directed mutagenesis, the host cells produced the ter-binding protein (Ter protein) over 2,000-fold. Using these over-producing cells as the enzyme source, the Ter protein was purified to apparent homogeneity. Molecular mass 36,000, amino-terminal amino acid sequence (45 residues) and composition of the protein were in good agreement with those deduced from DNA sequence of the tau gene. Footprinting using the purified Ter protein revealed a specific binding to the ter sequences.