FBL-3 lymphoma cells, originating in Friend leukemia virus recipient C57BL/6 mice injected at birth with the leukemia virus, induce a transient solid tumor when injected subcutaneously into syngeneic mice or a rapidly progressing tumor when injected i.p. Spleen cells from mice bearing the tumor show an impaired immune response to sheep erythrocytes. Incubation of spleen cells from normal syngeneic mice with FBL-3 cells markedly reduced the expected antibody response to sheep red cells in a dose-related manner. Addition of tumor cells to normal spleen cells as late as 72 hr after culture initiation still inhibited antibody formation. In contrast, cell-free homogenates derived from these tumor cells did not cause significant depression of antibody formation, as occurred with the inducing leukemia virus per se. Separation of FBL-3 cells from target splenocytes by cell-impermeable membranes prevented immunosuppression. Irradiation of the tumor cells or heating at 56°C or more inhibited the immunosuppressive activity of the tumor cells. Thus, immunosuppression induced in vitro by FBL-3 cells is not a function of a nutritional deficiency induced by the tumor cells in vitro or by a soluble factor secreted by viable tumor cells; contact with functional FBL-3 cells is necessary for impairment of the normal immune function of splenocytes co-cultured with the tumor cells.