The components for termination of DNA replication in Escherichia coli include the terminator signals T1 and T2 and the trans-acting gene tus. We have shown previously that tus maps in a 4-kilobase region of the chromosomal terminus near T2. Through the use of deletion and insertion mutants, the location of the tus gene has now been precisely identified. We sequenced 2416 nucleotides in this region and identified a 927-base-pair open reading frame which encodes Tus. Insertion of a kanamycin-resistance gene in this open reading frame abolished tus activity. We also demonstrated that crude extracts of tus+ cells contain a protein which binds to the T2 terminator sequence.