Donor-specific allograft tolerance can be induced in the adult rat by pregraft donor-specific blood transfusion (DST). This tolerance appeared to be mediated by regulatory cells and to the production of the suppressive cytokine TGF-beta1. A potential immunoregulatory CD8+ clone bearing a Vbeta18-Dbeta1-Jbeta2.7 TCR gene rearrangement was previously identified in DST-treated recipients. To assess the functional role of this T cell clone in the induction of tolerance by DST, we have vaccinated DST-treated recipients with a plasmid construct encoding for the Vbeta18-Dbeta1-Jbeta2.7 TCR beta-chain. DST-induced allograft tolerance was abolished by anti-TCR Vbeta18-Dbeta1-Jbeta2.7 DNA vaccination in six of seven recipients, whereas vaccination with the vector alone, or with the construct encoding a TCR Vbeta13 beta-chain, had no effect. However, the transcript number of the Vbeta18-Dbeta1-Jbeta2.7 chain was unchanged in allografts from vaccinated DST-treated rats, suggesting that this clone was not depleted by vaccination, but rather was altered in its function. Moreover, TCR Vbeta18-Dbeta1-Jbeta2.7 DNA vaccination restored the anti-donor alloantibody production, partially restore the capacity of spleen cells from tolerized recipients to proliferate in vitro against donor cells, and decreased the inhibitory effect of TGF-beta1, seen in DST-treated recipients, in spleen cells from vaccinated DST-treated ones. This study strongly suggests that this CD8+ TCR Vbeta18-Dbeta1-Jbeta2.7 T cell clone has an effective immunoregulatory function in allograft tolerance induced by DST.