Mixtures of isogeneic lymph node cells (LNC) and thymocytes (TC) exhibit far greater responsiveness in the murine MLI, as measured by proliferation and development of cytotoxic effector cells, than either cell type cultured alone. Pretreatment of either lnc or TC with mitomycin-C or ultraviolet irradiation completely abolished their synergistic interaction. Administration of cortisone acetate to cell donors 20 hr before sacrifice reduced the capacity of LNC and enhanced the capacity of TC to synergize. The LNC and TC populations participating in synergy, were found to be thymus dependent. LNC were shown to be responsible for the bulk of proliferative and effector activity observed in synergizing cultures, whereas TC appeared to amplify the activation of LNC. These findings provide the basis for a three cell model of MLI responsiveness.