The immune response to bovine insulin (BI) in the rat is controlled by the major histocompatibility complex (Mhc)-linked immune response gene (Ir-B1) and immune suppression gene (Is-B1). In the present study, we investigated the low responsiveness to BI in the WKAH rat (RT1 k) and attempted to explore the functional link between Is-BI and Mhc class II molecules. Lymph node cells (LNC) from the low responder (WKAH) rats responded well to BI when a large amount of antigen was added to the culture in vitro or after OX8-bearing (OX8 Γ·) T cells were eliminated. These LNC, after the elimination of OX8 Γ· cells, could show the RT1.Dk-restricted proliferative response upon in vitro challenge with BI, BI-B chain, or pork insulin. In addition, OX8 Γ· T cells, which were activated with BI and antigen-presenting cells (APC) in vitro, suppressed the anti-BI response of W3/25-bearing proliferating T cells from BI-immunized rats. The results have demonstrated that proliferating Tcell repertoires do exist to BI, which recognize BI-B chain in the context of RT1.D k molecules in the WKAH rat, and that the state of low responsiveness is mediated to a great extent by antigen-specific OX8 Γ· suppressor T (Ts) cells. Furthermore, the elimination of APC or the addition to RT1.Bk-specific monoclonal antibody in the in vitro secondary activation culture of Ts cells diminished the suppressive activity of OX8 Γ· Ts cells. In the induction phase of Ts cells it therefore seems to be necessary for these cells to recognize BI together with RT1.B k molecules on APC.