We recently described a series of phenolic thieno [2,3-d]pyrimidines, such as 1 (Figure ), that bind and activate the estrogen receptor a (ERa). These nonsteroidal estrogens were able to mimic the natural steroidal hormone 17b-estradiol (E2) with nanomolar affinities for ERa and potent agonist activity in reporter gene assays. Surprisingly, the X-ray cocrystal structure of thieno [2,3-d]pyrimidine (1) bound to ERa showed only a single hydrogen bond between the phenolic oxygen and E353 of the receptor (see Figure ). In contrast the phenolic oxygen of E2 forms an intricate network of hydrogen bonds with E353, R394, and a water molecule in the ERa ligand pocket that is essential for molecular recognition of steroidal estrogens. Initial analysis of the binding site suggested that the isomeric meta-phenol (2) would be an improved estrogen mimetic, however it was shown to be much weaker for both receptor binding and agonist activity. The result was especially surprising since the 2-isomer of the steroidal estrogen (Figure ) that places a phenol in the same position in the ligand binding pocket as para-phenol (1) has been shown to have a 5-fold weaker affinity for ERa. To resolve this puzzle and to further our understanding of how thieno [2,3-d]pyrimidines function as nonsteroidal estrogens we performed a series of structure-function, physiochemical, and computational studies to probe the interaction of the phenol pharmacophore with the receptor.