The DNA replication terminus (terR) of the R6K plasmid located on a 216 bp Alul fragment (Alu216) can block progress of the DNA replication fork. We previously developed an electrophoresis assay that allows detection of terminus activity on any DNA fragment cloned in the pUC vector. For precise identification of terR, we tested Alu216, its subfragments, and synthetic oligonucleotides by this assay. We found terR to be composed of a pair of separable sites, terR1 and terR2, each of which can block the DNA replication fork traveling in a specific but not the opposite direction. Both terR sites were composed of 22 nucleotides containing the repeated 20 bp sequence 5'-TAGTTACAACAC(A or T) CAA(G or T) AGA-3', located 73 bp apart in the inverted position of Alu216. A DNA homology search suggested that the R6K plasmid and the E. coli chromosome share a common termination system.