Two steroid binding states of an estrogen receptor each with different equilibrium constants (Kd values) Rx (Kd = 0.06 nM) and Ry (Kd = 0.8 nM) have been identified and characterized in the hen and estrogen-stimulated chick oviduct. A third non-estrogen binding form of the receptor, designated Rnb, has also been identified. These three forms of the receptor are interconvertible and appear to have a common molecular weight of approx. 66,000 under denaturing conditions. Hydroxytamoxifen binds preferentially and with high affinity to Rx (Kd 0.03 nM) and the conversion of Rx to Ry which is mediated by gamma phosphoryl group of ATP is also inhibitable by hydroxytamoxifen. Thus receptor interconversion, which may have general application to hormone action, potentially explains agonist/antagonist activity. The conversion of the non-estrogen binding form of the receptor (Rnb) to the lower affinity receptor (Ry) in chick oviduct cytosol is catalyzed by a reaction requiring the loss of the terminal phosphoryl moiety from ATP. There is a specific requirement for Mg2+. We now describe that ammonium sulfate fractionation of the cytosol allows the separation of the receptor entities from the "activating factor" (Fy) that catalyzes the conversion of Rnb to Ry. In the presence of gamma [32P]-ATP at 30 degrees C the purified non-steroid binding form of the receptor is phosphorylated. Phosphoamino acid analysis using Partisil-10 SAX anion exchange resin demonstrates that a serine is phosphorylated; and quantitation of the phosphorylation is indicative of one phosphoserine/receptor molecule. Treatment of the receptor with the partially purified activating factor to induce estradiol binding causes a dramatic reduction in phosphorylation.