A structural characteristic of many antiestrogens (AEs) is a bulky side chain with basic or polar functional groups that may interact with charged and polar amino acids near the hormone-binding site in the estrogen receptor. Recently, we have identified Cys530 as the residue of the human estrogen receptor that is the site of covalent labeling by aziridine analogs of estrogens (Es) and AEs (Harlow, K. W., Smith, D. N., Katzenellenbogen, J. A., Greene, G. L., and Katzenellenbogen, B. S. (1989) J. Biol. Chem. 264, 17476-17485). Since the aziridine function is on the bulky side chain of these ligands, Cys530 must be at or near the site of those side chain interactions. To probe these interactions, we have, by site-directed mutagenesis, made mutant human estrogen receptors in which charged and polar amino acids near Cys530 are changed (Glu523 to Gln, Lys529 and Lys531 to Gln, Asn532 to Asp, and Asp538 to Asn) so as to alter charge with minimal steric alteration. These receptors were expressed in mammalian (Chinese hamster ovary or COS-1) cells and assayed for their binding affinity for Es and AEs, their interaction with estrogen-responsive element DNA, and their ability to activate or suppress transcription of estrogen-responsive reporter genes. Two of the estrogen receptor mutants, KKN-QQD (mutation of Lys529, Lys531, and Asn532 to Gln529, Gln531, and Asp532, respectively) and KK-QQ (mutation of Lys529 and Lys531 to Gln529 and Gln531, respectively), in which the local charge is changed from +2 to -1 or 0, respectively, display an affinity for estradiol (E2) 5-10 times lower than that of the wild-type receptor, which is attributable to an enhanced rate of E2 dissociation. Although these mutant receptors have reduced affinity for a variety of Es, they retain unaltered affinity for AE. The profiles of transcriptional activation of reporter genes by various concentrations of E2 show that these two mutants (KKN-QQD and KK-QQ) require 40- and 15-fold higher E2 concentrations, respectively, to achieve half-maximal activity. In contrast, mutants E523Q and D538N, with changes at amino acids further from Cys530, were unaltered in their hormone binding and transactivation activity by E or AE. Interestingly, the AEs 4-hydroxytamoxifen, LY 117018, U 23469M, and ICI 164384 were 15-30-fold more effective in inhibiting E2-stimulated transcription by mutants KKN-QQD and KK-QQ compared to the wild-type receptor.(ABSTRACT TRUNCATED AT 400 WORDS)