Infection of human lymphocytes with Epstein Barr virus (EBV) activates the release of lymphokines. Previous experiments have emphasized the ability of interferon-gamma (IFN-gamma) to prevent EBV-induced B cell transformation. However, the factors that regulate IFN-gamma synthesis and release during in vitro EBV infection are controversial. In the present investigation we have systematically evaluated the kinetics of production, cellular origins, and accessory cell requirements for IFN-alpha and IFN-gamma and for IL 1 and IL 2, after EBV infection. Our data indicate that IFN-alpha is released entirely by natural killer (NK) cells and B cells, in the absence of accessory cells, independently of the other lymphokines and within 24 hr of infection. In contradistinction, IFN-gamma secretion is exclusively of T cell origin, is absolutely dependent on the prior elaboration of IL 1 and IL 2, and is maximal 8 days after EBV infection. IL 2 secretion by T cells peaks on day 5 and requires the earlier release of IL 1. Both NK cells and monocytes are a source of IL 1. Secretion of IL 2 and IFN-gamma occurs in the presence of either one of these cell types but not in the absence of both. Antibody against IL 1 blocks EBV-induced IL 2 and IFN-gamma generation, and antibody against IL 2 decreases production of IFN-gamma. Thus, the production of IFN-gamma, the lymphokine that prevents EBV-induced B cell transformation, is the final outcome of a cascade of lymphokine-mediated events that involve interactions between virus-infected B lymphocytes that serve as antigen-presenting cells, NK cells and monocytes as sources of IL 1, and T lymphoblasts. Dysfunctions of any or all of these cell types would be expected to impair the regulation of EBV transformation.