The role of macrophages in the production of two lymphokines, monocyte chemotactic factor and macrophage activating factor, was investigated. Lymphokine production by guinea pig lymph node and spleen cells required macrophages for thymus-dependent antigens and mitogens. In contrast, B cell stimulants which also induce the synthesis of lymphokines were macrophage independent. When populations of relatively pure B or T lymphocytes were isolated, it was found that T cells required viable macrophage cooperation to produce these two lymphokines and to undergo proliferation in response to specific antigens, whereas B cells could be directly activated in the absence of macrophages. These findings suggest that T and B cells have different requirements for activation and for macrophage cooperation. Furthermore, since lymphokine synthesis is evident within the first 4 hr of stimulant presentation, these observations demonstrate that macrophages play an essential role in the earliest events of lymphocyte activation.