We have studied the specific binding of 1,3,5(10)-estratrien-2,3,17 beta-triol (2-hydroxyestradiol) to an enriched membrane fraction isolated from the rat anterior pituitary gland. Specific [6,7-(3)H]2-hydroxyestradiol-17 beta ([3H]2-hydroxyestradiol) binding is saturable and displays a high and low affinity binding component. The apparent dissociation constants, KD, are 4 +/- 2 x 10(-10) and 2 x 10(-6) M with 13 fmol and 2.6 pmol bound/mg of protein, respectively. The specific high affinity binding increases linearly with increasing amounts of tissue protein. The binding is both temperature- and pH-dependent, with maximal binding at 37 degrees C and pH 7.4. The 2-hydroxyestradiol binding is shown to be stereospecific. Dopamine and spiroperidol (a dopamine antagonist) competitively inhibit the specific binding of [3H]2-hydroxyestradiol to the high affinity binding site with inhibition constants, KI, of 1 x 10(-6) M and 2 x 10(-5) M, respectively. Related catecholestrogens (2-hydroxyestrone and 2-hydroxyestriol) are also competitive inhibitors of [3H]2-hydroxyestradiol binding with KI values of 1.5 x 10(-5) M and 1.9 x 10(-5) M, respectively. None of the estrogens (estrone, estradiol, estriol) or 2-methoxyestrogen derivatives (2-methoxyestrone, 2-methoxyestradiol, 2-methoxyestriol) inhibits [3H]2-hydroxyestradiol binding at concentrations up to 10(-4) M. Norepinephrine, epinephrine, and serotonin are also ineffective as inhibitors at concentrations of 10(-5) M and inhibited less than 20% at 10(-4) M. Centrifugation through a stepwise discontinuous sucrose density gradient is used to separate subcellular components of the anterior pituitary cells. Approximately 90 per cent of the specific [3H]2-hydroxyestradiol binding is associated with the material at the 47.4 to 52.9% sucrose interface, the layer most highly enriched for 5'-nucleotidase (an enzyme marker for plasma membranes). Studies of tissue specificity indicate that specific 2-hydroxyestradiol binding sites are heterogeneously distributed in nervous tissue with the highest concentration of binding sites in the anterior pituitary, cerebral cortex, and hypothalamus and lower levels found in the thalamus and striatum. Low levels 2-hydroxyestradiol binding sites are also identified in liver and uterus. The present demonstration of specific 2-hydroxyestradiol binding to the anterior pituitary membrane provides information on the mechanism of catecholestrogen action.