Ficoll-purified lymphocytes (peritoneal, splenic, or thymic) and macrophages (peritoneal) from Toxoplasma-immune and normal female NMRI mice were used. Suspensions of washed cells were made in medium 199 containing 20% heat-inactivated normal calf serum. Sixty minutes after the adherence of 10(5) macrophages to cover slips in Leighton tubes, lymphocytes were added in various concentrations. The mixed cellular population was then incubated at 37 C. Eighteen hours later, most of the lymphocytes were firmly attached to macrophages to form rosettes. This cellular interaction, which was temperature, cell ratio, and time dependent, occurred in the absence of any particular antigenic stimulation. Morever, the reaction was cytotoxic only for adhered lymphocytes as judged by staining with 0.2% trypan blue. Splenic and thymic lymphocytes were bound in significantly greater number than peritoneal lymphocytes. Incubation of macrophages for more than 48 h at 37 C before the addition of fresh lymphocytes markedly reduced rosette formation. Treatment of macrophages and lymphocytes with mouse anti-immunoglobulin did not affect the reaction. The labeling of lymphocytes with fluorescent anti-mouse sera and the use of nude NMRI mice showed that both B and T cells can form spontaneous rosettes with syngeneic peritoneal macrophages.