Multicellular spheroids of EMT6 mammary sarcoma cells of BALB/c origin were incubated with normal spleen cells or alloimmune spleen cells generated in vitro in mixed leukocyte cultures (MLC). After 24 hours, spheroids were trypsinized and assayed for surviving tumor cells by use of a cloning technique. Under these conditions a 60-80% reduction in clone-forming tumor cells was observed after incubation of spheroids with immune lymphocytes as compared to normal lymphocyte controls. This cytotoxic effect occurred in situ, and alloimmune cells sensitized against unrelated antigens were much less cytotoxic than were specifically sensitized cells. In parallel autoradiographic studies, some immune lymphoid cells that had been labeled with tritiated thymidine during the proliferative phase of the MLC could be demonstrated within spheroids after 24 hours. These results suggested that multicellular spheroids will be a useful in vitro model for more detailed analysis of the factors controlling infiltration in in situ destruction of solid tumor grafts.