The objective of the present study was to determine the ability of 17β-estradiol (E₂) and conjugated forms of E₂ (E₂ conjugated to BSA [E₂-BSA] and a novel conjugate, E₂ conjugated to a small peptide [E₂-PEP]) to prevent the GnRH-induced secretion of LH and to determine the role of estradiol receptors (ERs) and ER subtypes (ERα, also known as ESR1, and ERβ, also known as ESR2) in the mediation of the acute action of E₂ in primary cultures of ovine pituitary cells. Preincubation of cells for 15 min with E₂, E₂-BSA, or E₂-PEP prevented the GnRH-induced secretion of LH (P < 0.01). Treatment of cells with nonestrogenic steroid hormones did not affect secretion of LH when given alone, nor did these steroids impair the E₂-induced inhibition of LH secretion (P > 0.1). Likewise, treatment of cells with the ER-antagonists tamoxifen, hydroxytamoxifen, or ICI 182 780 did not affect (P > 0.1) secretion of LH when given alone but did prevent (P < 0.01) the inhibition by E₂ and the E₂-conjugates on GnRH-induced secretion of LH. When cells were treated with subtype-selective ER agonists, the ERα agonist (propylpyrazole-triol), but not the ERβ agonist (diarylpropionitrile), decreased (P < 0.01) the GnRH-induced secretion of LH. In conclusion, the rapidity by which E₂ prevented GnRH-induced release of LH in ovine pituitary cells suggests that this inhibition is mediated via a nongenomic action of E₂. The inhibition of GnRH-induced secretion of LH proved to be steroid specific and mediated by ERs. It may occur specifically through ERα. The fact that E₂-BSA or E₂-PEP mimicked the action of E₂ suggests that this effect was mediated by an ER associated with the plasma membrane.