The over-expressing groEx gene of symbiotic X-bacteria in Amoeba proteus has unique nucleotide motifs (Tx), containing two hairpins and a C-rich region at its 3'-end. To investigate the role of Tx as a transcription terminator, we mutated Tx and analyzed the effects on the expression of an upstream-located lacZ in E. coli. The level of beta-galactosidase (beta-gal) production in Tx deletion mutants was reduced to 23% of the control level. Site-directed mutation of the hairpin-1, C-rich region, and hairpin-2 reduced the beta-gal production to 28-64%, 33, and 20% of wild-type Tx, respectively. The amount of lacZ transcripts that were detected in RNA blots was proportional to the level of beta-gal. The Tx sequence had 97% termination efficiency in vivo, and the mutations in Tx resulted in read-through transcripts with significantly shortened half-life. In rho- E. coli, the level of the beta-gal production by Tx was reduced to 15% of that in rho+ E. coli. These results suggest that Tx is a Rho-dependent transcription terminator. Also, hairpin-2 is the most effective component among the three motifs of Tx for proper termination of the transcription and stability of mRNAs.