The graft-vs-host (GVH) activity of thymocytes from BALB/c mice treated 48 to 72 hr previously with 2.5 mg cortisone acetate has been studied with both a spleen weight assay and a mortality assay. Although the cortisone-resistant (CR) thymocytes were 10 times as active as normal thymocytes in the spleen weight assay, they were only 4 to 5 times as active in the mortality assay, and the yield of thymus cells from cortisone-treated donors was only 1/25 that from normal donors: such pretreatment thus resulted in over 50% loss of total GVH reactivity recoverable from the thymus when measured by the spleen weight assay, and approximately 80% loss when measured by the mortality assay. In contrast to normal thymocytes, CR-thymocytes were unable to interact synergistically with normal peripheral lymph node cells; they were also unable to interact synergistically with normal thymocytes. The kinetics of cumulative mortality induced by CR-thymocytes were identical to those of normal thymocytes and strikingly different from those produced by various peripheral lymphoid cell populations. These results suggest that CR-thymocytes represent a unique population of thymus-derived cells and lend support to the concept that different expressions of cell-mediated immune reactions may be initiated by distinct cell types.