SUMMARYAn in vitro graft reaction system in which rat lymphocytes were sensitized in culture against mouse fibroblasts, then were tested for their capacity to effect immune lysis of mouse cells, was found to be based on a bicellular lymphoid interaction. Rat thymus cells, which by themselves are incapable of effecting the lytic reaction following sensitization on mouse fibroblasts, when combined with spleen cells, confer specific lytic activity on the mixed cell population. Experiments were made to define the tissue origin and properties of the spleen cells which are capable of interacting with the thymus cells. A mixed population of bone marrow and thymus cells, combined and cultured on mouse cells, did not manifest immune lysis of mouse target cells. Mixed populations consisting of spleen cells of thymectomized, X-irradiated mice inoculated with rat marrow and of rat thymus cells combined and cultured on mouse fibroblasts did show lytic activity. Thus, bone marrowderived cells within the spleen interact with thymus cells to effect lysis of target monolayers. The capacity of such bone marrow cells to recognize transplantation antigens was studied. Rat bone marrow-derived cells (following colonization of B6D2F1 mouse spleens), when admixed with rat thymus cells, could be sensitized and would effect lytic activity on C3H, but not on C57BL and DBA/2 strain, fibroblasts. Thus, the rat bone marrow-derived cells seemed to recognize the recipient's mouse antigen, since they manifested unresponsiveness toward fibroblasts of similar antigenic constitution. We conclude that: (1) the in vitro graft reaction system is based on interaction between thymusprocessed and nonthymus-processed lymphocytes; (2) the nonthymus-processed cells derive from the bone marrow but are not identical with the original bone marrow cells. They seem to represent a differentiation product of bone marrow cells; (3) both the thymus- and the nonthymus-processed cells are capable of recognizing the mouse antigens. The question of which is the killer cell remains open. The necessary participation of thymus cells in the killing process is discussed