We have systematically evaluated the effect of trislphosphate buffers, ethylenediamine tetraacetate (EDTA), dithiothreitol (DTT), molybdate, and phenylmethylsulfonyl fluoride (PMSF) on the binding of 170estradiol to human BPH cytosol. Competition binding assays demonstrated that the estrogen receptor was highly specific for 170-estradiol. There was statistically no difference in binding observed with tris or phosphate buffers. Two classes of binding (high affinity-type I, low affinity-type II) were observed. DTT (1 mM) showed maximum inhibition of type II binding. There was no significant difference in binding observed in the presence of 1.0 mM EDTA. However, 10 mM EDTA stimulated estradiol binding in both saturation analysis and glycerol gradients. Stimulation of estradiol binding by 10 mM EDTA was ob-served even in the presence of 1 mM DTT. Molybdate (20 mM), a reagent used to stabilize steroid receptors, significantly inhibited estradiol binding. PMSF inhibited estradiol binding at all concentrations studied and the nature of the inhibition appeared to be uncompetitive. A B , , , (maximum number of binding sites) of 84.4 * 68.5 fmoleslmg protein with a KD (equilibrium dissociation constant) of 2.5 f 1.7 mM was observed for type I estrogen receptor binding. Our B, , , values are higher than those reported in other studies even after allowing for possible overestimation. In view of the potential importance of estrogen receptors in human prostate, careful reevaluation of estrogen receptors must be undertaken using proper assay conditions.