To test the hypothesis that the putative 89K oriT sequence plays a crucial role in the initiation of conjugal transfer, a 355 bp segment containing the putative oriT site was cloned into a non-transmissible Escherichia coli-S. suis shuttle plasmid, pVA838. The recombinant plasmid (pVA838-oriT) was found to be able to transfer at a frequency of about 6.1 ¥ 10 -4 transconjugants per donor, which is approximately two orders of magnitude greater than that of 89K*. PCR analysis confirmed the efficient mobilization of pVA838-oriT by using specific primers. However, no transconjugants were obtained when the DmobA89K mutant was used as the carrier donor. These results demonstrated that the nonconjugative plasmid pVA838 could be mobilized only by 05ZYH33 carrying an active relaxase after introducing a fragment encompassing the putative 89K oriT. In other words, the 89K PAI has a functional oriT site recognized by its cognate relaxase, MobA89K.