The pituitary hormone prolactin (PRL) is also produced by cells of the immune system and participates in early and late T cell activating events. We have previously shown a modulatory role of PRL during maturation of dendritic cells (DC). Production of IL-12 by T cell receptor (TCR)-activated DC is necessary for T cells to acquire the Th1 cytokine (i.e., IFN-gamma secreting) profile, which is associated with activation of cellular response. In a separate work, PRL has been shown to increase IFN-gamma synthesis by natural killer (NK) cells. We have extended that study by exploring the ability of PRL to induce IFN-gamma production by T and NK cells in the presence of the specific stimuli IL-12 and IL-2. The individual effect of PRL, IL-12, and IL-2 was specific for NK cells, and IL-2 and IL-12 were much more efficient than PRL. Cooperation of IL-2 and PRL was observed on NK cells. IL-2-induced synthesis of IFN-gamma was increased by physiological concentrations of PRL but was unaffected or inhibited by high concentrations. By contrast, optimal enhancement of IL-12-induced IFN-gamma release was observed with T cells but not with NK cells. Unexpectedly, interaction between PRL and IL-12 occurred only at high concentrations of PRL. These data indicate a complex role of PRL in the cytokine network and point to a revaluation of the proposed immunosuppression by stress-related hyperprolactinemia.