The goal of these studies was to define the stimuli and factors that control the induction of anergy in unimmunized resting T lymphocytes. Initial experiments, aimed at establishing the system, showed that exposure of Th1 but not Th2 clones to immobilized anti-CD3 leads to a block in autocrine growth factor production and proliferation upon subsequent restimulation with Ag+APC. Anergy is not prevented by accessory cells, suggesting that this model of T cell tolerance may be due to receptor-mediated inhibitory signals, independent of costimulatory molecules. Culture of small (resting) unimmunized T lymphocytes with anti-CD3 +/- IL-2 induces unresponsiveness to restimulation with anti-CD3, but culture with anti-CD3+IL-4, which stimulates the differentiation of resting cells into IL-4 producers, does not induce anergy. Thus, IL-4-producing clones and bulk populations of IL-4-producing T cells are resistant to Ag receptor-mediated inhibitory stimuli. These results provide experimental models for studying the mechanisms of anergy in normal, unselected, mature T cells, and demonstrate fundamental similarities between cloned cell lines and unimmunized T lymphocytes in the induction of anergy.