To search for endogenous estrogens that may have preferential binding affinity for human estrogen receptor (ER) or subtype, and also to gain insights into the structural determinants favoring differential subtype binding, we studied the binding affinities of 74 natural or synthetic estrogens (including >50 steroidal analogs of estradiol-17 [E₂] and estrone [E₁]) for human ER and ER. Many of the endogenous estrogen metabolites retained varying degrees of similar binding affinity for ER and ER, but some of them retained differential binding affinity for the two subtypes. For instance, several of the D-ring metabolites, such as 16-OH-E₂, 16-OH-E₂-17, and 16-keto-E₁, had distinct, preferential binding affinity for human ER over ER (difference up to 18-fold). Notably, while E₂ has nearly the highest and equal binding affinity for ER and ER, E₁ and 2-OH-E₁ (two quantitatively-predominant endogenous estrogens in nonpregnant woman) have preferential binding affinity for ER over ER, whereas 16-OH-E₂ (estriol) and other D-ring metabolites (quantitatively-predominant endogenous estrogens formed during pregnancy) have preferential binding affinity for ER over ER. Hence, facile metabolic conversion of parent hormone E₂ to various metabolites under different physiological conditions may serve unique functions by providing differential activation of the ER or ER signaling system. Lastly, our computational 3D-QSAR/CoMFA analysis of 47 steroidal estrogen analogs for human ER and ER yielded useful information on the structural features that determine the preferential activation of the ER and ER subtypes, which may aid in the rational design of selective ligands for each human ER subtype.