The molecular mechanisms of activation of immunoregulatory T cells were characterized by using two complementary suppressor T cell hybridoma systems: the KLH-specific monoclonal suppressor factor (KLH-TsF), and the inducible acceptor-suppressor hybridoma line with anti-idiotypic receptor for KLH-TsF. It was demonstrated that the identity of the KLH specificity and genetic specificity was required for the TsF-acceptor interaction. These specificities were found to be mediated by the two polypeptide chains of TsF: KLH-binding, Ct-bearing heavy chain and I-J+ light chain. These two chains were essential for stimulation of the acceptor hybridoma. The results were also confirmed by the findings that the mixture of the 11S and 13S mRNA translation products reconstituted the active TsF to stimulate the acceptor hybridoma. Furthermore, the genetic restriction observed was found to be mediated by the I-J+ light chain and to be governed by the gene linked to the H-2 complex but not to the Igh genes. The gene controlling the restriction specificity was strongly suggested to be in the intra-H-2 complex, but not outside of the H-2 complex.