The ability of murine spleen, lymph node, but not thymus cells, cultured in the presence of heterologous serum to suppress normal spleen cells from responding in a cell-mediated cytotoxicity (CMC) generation culture are described. That the suppressor cells were T cells was demonstrated by their sensitivity to anti-θ serum and complement. Suppression was totally abolished by relatively low doses (250 R) of irradiation prior to their addition to test CMC generation cultures. The culture conditions necessary for suppressor generation are described. Delayed addition of suppressors to CMC generation cultures was carried out and demonstrated that the suppressor effect was operative on an early phase of allogeneic recognition and activation. If suppressor cells were added after 72 hr of CMC generation culture, no suppression could be demonstrated. Effector function (i.e., cytotoxicity) was not influenced by suppressor cells. Additional functional capabilities of the suppressor population were tested. They were found to be unable to generate CMC in response to allogeneic spleen cells. They were, however, responsive to both T (canavalin A) and B (lipopolysaccharide) cell mitogens. In addition, the high spontaneous thymidine uptake of the suppressor population suggested that the suppressor cell may well be a stimulated T lymphoblast. Separation of suppressor cells from the test CMC generation cultures in a modified Marbrook system could not demonstrate the presence of a soluble factor mediating this suppression.