This study assessed the importance of cortisol in mediating inhibition of pulsatile LH secretion in sheep exposed to a psychosocial stress. First, we developed an acute psychosocial stress model that involves sequential layering of novel stressors over 3-4 h. This layered-stress paradigm robustly activated the hypothalamic-pituitary-adrenal axis and unambiguously inhibited pulsatile LH secretion. We next used this paradigm to test the hypothesis that cortisol, acting via the type II glucocorticoid receptor (GR), mediates stress-induced suppression of pulsatile LH secretion. Our approach was to determine whether an antagonist of the type II GR (RU486) reverses inhibition of LH pulsatility in response to the layered stress. We used two animal models to assess different aspects of LH pulse regulation. With the first model (ovariectomized ewe), LH pulse characteristics could vary as a function of both altered GnRH pulses and pituitary responsiveness to GnRH. In this case, antagonism of the type II GR did not prevent stress-induced inhibition of pulsatile LH secretion. With the second model (pituitary-clamped ovariectomized ewe), pulsatile GnRH input to the pituitary was fixed to enable assessment of stress effects specifically at the pituitary level. In this case, the layered stress inhibited pituitary responsiveness to GnRH and antagonism of the type II GR reversed the effect. Collectively, these findings indicate acute psychosocial stress inhibits pulsatile LH secretion, at least in part, by reducing pituitary responsiveness to GnRH. Cortisol, acting via the type II GR, is an obligatory mediator of this effect. However, under conditions in which GnRH input to the pituitary is not clamped, antagonism of the type II GR does not prevent stress-induced inhibition of LH pulsatility, implicating an additional pathway of suppression that is independent of cortisol acting via this receptor.