In the rat, it is generally accepted that the primary site of estrogen's stimulatory (positive) effects on serum LH is the preoptic area-anterior hypothalamus (POA/ AH). In contrast, the primary site of estrogen's inhibitory (negative) effects on serum LH levels has not been conclusively identified. There is evidence to suggest both a medial basal hypothalamic (MBH) and an anterior pituitary site of action. The present studies utilized a unique characteristic of these estrogen effects to investigate their putative loci. Extensive dose-response curves of estrogen's induction of positive and negative feedback indicated that the negative feedback response was activated at a lower concentration of serum estradiol than the positive feedback response. The differential sensitivities of these two responses suggested that the tissues mediating them might also be differentially sensitive to estradiol. In a previous paper, we showed that receptor translocation is an index of estrogen sensitivity. We measured receptor translocation in response to a series of estradiol doses in the POA/AH, the MBH, and the pituitary. Dose-response curves for estrogen's effect on receptor translocation showed that the pituitary receptor translocation mechanism is activated at significantly lower levels of serum estradiol than that of either the POA/AH or the MBH. These results are consistent with the POA/AH as a site of estrogen's positive feedback effects. In addition, they suggest that negative feedback in the rat may be mediated via estrogen's action at the anterior pituitary. Estrogen's negative feedback effect on serum LH occurs at a serum level of estrogen at which no receptor is translocated in the MBH. Therefore, the pituitary, which does possess nuclear receptors at these estradiol dose levels, is more likely to be the primary mediator of estrogen's negative feedback effects. In another experiment, pituitary, but not hypothalamic, receptor was translocated to the nuclear fraction with an injection of 100 pg clomiphere