SUMMARYCell-free medium (CFM) from cultures of unmixed or mixed irradiated human leukocytes stimulated3H-thymidine uptake by fresh allogeneic leukocytes. At least some component of the blastogenic factor was antigenically specific. The blastogenic factor from cultures of irradiated leukocyte mixtures was much more active than that from cultures of unmixed irradiated leukocytes. The activity of the blastogenic factor was affected by the dose of irradiation and the time interval between the end of irradiation and the preparation of cultures. The blastogenic factor from mixed cultures of irradiated and intact cells was consistently less stimulatory to fresh leukocytes from the donor of the irradiated cells in the original mixture (source of CFM) than it was to fresh leukocytes from the original donor of the intact cells. These data raise the possibility that production of a blastogenic factor may be involved in the stimulation of responding lymphocytes in mixed cultures.