Stability of plant tumor-inducing (Ti) plasmids differs among strains. A high level of stability prevents basic and applied studies including the development of useful strains. The nopaline type Ti plasmid pTiC58 significantly reduces the transconjugant efficiency for incoming incompatible plasmids relative to the other type, such as octopine-type plasmids. In this study we identified a region that increases the incompatibility and stability of the plasmid. This region was located on a 4.3-kbp segment about 38 kbp downstream of the replication locus, repABC. We named two open reading frames in the segment, ietA and ietS, both of which were essential for the high level of incompatibility and stability. Plasmid stabilization by ietAS was accomplished by a toxin-antitoxin (TA) mechanism, where IetS is the toxin and IetA is the antitoxin. A database search revealed that putative IetA and IetS proteins are highly similar to AAA-ATPases and subtilisin-like serine proteases, respectively. Amino acid substitution experiments in each of the highly conserved characteristic residues, in both putative enzymes, suggested that the protease activity is essential and that ATP binding activity is important for the operation of the TA system. The ietAS-containing repABC plasmids expelled Ti plasmids even in strains which were tolerant to conventional Ti-curing treatments.