The Escherichia coli replication terminator TerB was inserted in its two alternate orientations into a Bacillus subtilis fork-arrest assay plasmid. After transferring these new plasmids into B. subtilis, which could overproduce the E. coli terminator protein Tus, it was shown that the E. coli Tus-TerB complex could cause polar replication fork arrest, albeit at a very low level, in B. subtilis. A new B. subtilis-E. coli shuttle plasmid was designed to allow the insertion of either the Terl (B. subtilis) or TerB (E. coli) terminator at the same site and in the active orientation in relation to the approaching replication fork generated in either organism. Fork-arrest assays for both terminator-containing plasmids replicating in both organisms which also produced saturating levels of either the B. subtilis terminator protein (RTP) or Tus were performed. The efficiency of the Tus-TerB complex in causing fork arrest was much higher in E. coli than in B. subtilis. The efficiency of the B. subtilis RTP-Terl complex was higher in B. subtilis than in E. coli, but the effect was significantly less. Evidently a specificity feature in E. coli operates to enhance appreciably the fork-arrest efficiency of a Tus-Ter complex. The specificity effect is of less significance for an RTP-Ter complex functioning in B. subtilis.