We have previously reported that two types of suppressor T cell factors (TsF) specific for L-glutamic acid50-L-tyrosine50 (GT) or L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT) can be distinguished based upon differences in their ability to suppress responses by allogeneic mice. Injection of GAT or GT induces a suppressor T cell subset that produces an antigen-binding, I-J+, genetically unrestricted, specific suppressor factor (TsF1). Injection of this factor plus small amounts of antigen induces a second-order suppressor T cell that produces an antigen-binding, I-J+, genetically restricted, specific suppressor factor (TsF2). In this report, we demonstrate that these two factors are also biochemically distinct. Monoclonal TsF1 molecules are composed of a single polypeptide chain that bears both the antigen-binding site and I-J determinant, whereas TsF2 molecules are composed of two disulfide-linked polypeptide chains, one of which is antigen-binding and I-J-, and the other, nonantigen-binding, I-J+. The antigen-binding chain must be added at culture initiation to achieve suppression, but the I-J+ chain can be added as late as day 3 with complete suppression observed. However, isolated chains from TsF2-producing hybridomas derived from three different haplotypes were unable to suppress immune responses when chains from heterologous TsF2 were mixed. Indirect evidence is presented that suggests that this restriction is because the chains fail to interact rather than the inability of the target cells to recognize both chains.