A comparison was made between two procedures which give rise to in vitro activation of normal mouse peritoneal macrophages: (1) normal macrophages were incubated for 22 h with sensitized lymphocytes and antigen (assay A), and (2) normal macrophages were incubated for 70 h with supernatants from sensitized lymphocytes and antigen (assay B). The activation of macrophages was measured as an increase in 1-14 C glucose oxidation. Combinations of lymphocytes and macrophages is probably necessary in assay A.s from different mouse strains demonstrated that activation of macrophages in assay A, but not in assay B, required cells which were derived from strains of mice sharing identical H-2 antigens. Treatment of lymphocytes with mitomycin C blocked the activation of macrophages in both assays. Addition of alpha-L-fucose (0.1 M) during the incubation period blocked the activation of macrophages in assay B, but not in assay A. It is concluded that there is a qualitative difference between the two methods for activating macrophages and that direct contact between lymphocytes and macrophages is probably necessary in assay A.