We have previously demonstrated that LHRH is capable of eliciting a dramatic and direct elevation of anterior pituitary levels of nuclear estrogen receptors (E2R) [18, 24]. This action was manifest under both in vitro and in vivo conditions, and could not be attributed to an amplification of the rate or extent of cytosol E2R binding, activation or transloca-tion. In view of the implications of these observations in terms of estrogen-LHRH interplay in regulation of gonadotropin secretion, we have further explored the nature, specificity and estrogen dependency of the LHRH effect. TRH, a long-acting agonist, (D-Ala<sup>6</sup>, des-Gly<sup>10</sup>)LHRH-N-ethylamide (LHRH-A), and an antagonist, N-Ac-D-p-Cl-Phe<sup>12</sup>, D-Trp<sup>3</sup>, D-Phe<sup>6</sup>, D-Ala<sup>10</sup>-LHRH (LHRH-ANT), were compared with LHRH for their ability to alter compartmental E2R concentrations following incubation in vitro. Either intact dispersed anterior pituitary cells or isolated cytosol and nuclei from pituitary homogenates were used as receptor source. LHRH-A and LHRH both caused a 40–60% decrease in the cytoplasmic E2R binding capacity of intact cells following incubation of 30 min at 37 °C; the magnitude of the inhibition was dose independent over a peptide range of 0.1–1000 pmol/pituitary. In contrast, TRH caused a dose-related decrease in cytosol E2R binding. When nuclear E2R levels were assessed, significant dose dependent increases were observed with both LHRH and LHRH-A treatment. The effective dose of LHRH-A was approximately an order of magnitude lower than that of LHRH. TRH, on the other hand, over a wide concentration range (0.3–3,000 pmol/pituitary), had no significant effect on specific nuclear estrogen binding. In isolated nuclei, as in the intact cell studies, LHRH and LHRH-A were stimulatory, while TRH effected no alteration of E2R binding levels. The LHRH effect could be totally blocked by LHRH-ANT. In isolated cytosol, TRH did not affect E2R binding, whereas LHRH and LHRH-A were both mildly stimulatory. The effectiveness of LHRH in enhancing nuclear E2R binding correlated directly with the endogenous estrogen titers of the animals. In tissue from ovariectomized animals primed with 0.1 μg estradiol/day, LHRH was 4 times more potent as a stimulator of nuclear E2R than in tissue from nonprimed castrate animals. If the priming dose was increased to 1.0 μg/day, a further significant increase in sensitivity of nuclear E2R to LHRH was observed over the 0.1 μg priming dose level. The results of these studies attest to the releasing hormone specificity of changes in anterior pituitary E2R levels, and to the involvement of estrogen-LHRH sensitization in this process, analogous to the well-documented estrogen-LHRH interplay in regulation of LHRH release.