Normal human T lymphocytes respond with vigorous proliferation when exposed in vitro to autologous irradiated lymphocytes enriched in B cells. In this report we examined the effect of various humoral factors that might play a role in the regulation of this autologous MLR. Neither fresh autologous plasma, thymic factors, nor hepatoma-derived α-fetoprotein had any significant suppressive effect on the autologous (or allogeneic) MLR. In contrast, over 98% suppression of the autologous MLR occurred with addition of 4 × 10-7 M hydrocortisone succinate. Interestingly, the presence of 2 to 8 × 10-8 M hydrocortisone during the autologous MLC suppressed 62 to 84% of the proliferation, whereas it did not suppress proliferation in the allogeneic MLC. Kinetic studies demonstrated that the predominant suppression of proliferation in the autologous MLC by hydrocortisone occurred during the first few days of culture and that by 100 hr of culture (which is before most of the DNA synthesis occurs) the addition or removal of hydrocortisone had no further effect on the reaction. The presence of hydrocortisone during just the first 6 hr of the autologous MLC also had no suppressive effect on proliferation. Thus, hydrocortisone alters critical functions at a cellular level without causing destruction or other irreversible effects on potentially autoreactive lymphocytes. Furthermore, marked suppression of the autologous MLR without any suppression of the allogeneic MLR was observed with concentrations of free cortisol equivalent to those found in human plasma, suggesting that hydrocortisone may have a physiologic role in the regulation of such T cell autoreactivity in vivo.