We have been interested in understanding how the estrogen receptor (ER) binds estrogens and discriminates between different classes of steroids with closely related structures. Using insights from our prior studies on ER and from sequence comparisons of steroid receptors, we identified three residues in the hormone-binding domain of the human ER, Leu345, Thr347, and Glu353, that we considered were likely to be involved in steroid A-ring recognition and therefore estrogen versus androgen discrimination. We then tested the effect on ER activity of mutating these ER residues to the corresponding androgen receptor residues. Specifically, we examined the ability of the mutant receptors to bind and be activated by 17beta-estradiol and three different androgens. No change in receptor activity was observed with the T347N mutation, while the L345S mutation greatly reduced ER activity in response to all ligands. Interestingly, the E353Q substitution behaved as expected, causing a 9-fold reduction in the transactivation potency of estradiol and a concomitant 10-140-fold increase in the transactivation potency of different androgens. These reciprocal changes in the transcriptional effectiveness of estrogens and androgens correlated with a decreased affinity of the E353Q ER for estradiol binding and an increased affinity for androgen binding. Therefore, amino acid Glu353 appears to be playing a significant role in binding the A-ring phenolic group of estradiol and in receptor discrimination between estrogens and the most closely structurally related steroids, androgens. Based on this data and our earlier observations, we propose a model for the orientation of ligand within the binding pocket of ER in which the A-ring 3-phenol of estradiol is hydrogen bonded to Glu353 in helix-3 and the 17beta-hydroxyl of estradiol is hydrogen bonded to His524 in helix-11. Our findings with estrogen and androgen suggest that this orientation of the steroid in the ligand-binding pocket, with the steroid A-ring in contact with helix-3 and the D-ring in contact with helix-11 residues, is likely to be general for all the steroid hormone receptors.