The previous communication presented data detailing that glucocorticoid hormones exhibited slight but significant inhibitory effects on the proliferation of cytolytic T cells maintained in long-term T cell growth factor (TCGF)-dependent cultures. However, most importantly, glucocorticoids were shown to inhibit T-lymphocyte proliferation via a profound effect on TCGF production. Addition of exogenous TCGF to glucocorticoid-treated human, mouse, or rat T cell mitogen-stimulated lymphocytes restored normal levels of proliferation. These observations led us to propose that the immunosuppressive effects of glucocorticoid hormones were mediated by controlling the production of the T cell proliferation-inducing agent, TCGF. Results of experimentation detailed in this communication provide further evidence in support of this hypothesis. We found that although glucocorticoid hormones had little effect on the cytolytic reactivity of T cells harvested from TCGF-dependent culture, treatment of mixed lymphocyte cultures (MLC) with identical glucocorticoid hormone concentrations completely abrogated in situ TCGF production and alloantigen-directed cytolysis. The inhibitory effects of glucocorticoids in MLC could be eliminated once again simply by the delayed addition of exogenous TCGF. These observations coupled with those detailed in the previous report provide evidence that, rather than effecting lysis of potentially reactive mature lymphocytes, glucocorticoids influence the development of T cell-mediated immune reactivity by inhibiting TCGF production, which in turn serves to inhibit the clonal expansion of activated T cells.