To better understand structure-activity relationships in the human estrogen receptor (ER), we examined the role of tyrosine 537 in the transcriptional response of the receptor, since this residue is close to a region of the hormone-binding domain shown previously to be important in hormone-dependent transcriptional activity and because this amino acid has been proposed to be a tyrosine kinase phosphorylation site important in the activity of the ER. We substituted five amino acids at this position (alanine, phenylalanine, glutamic acid, lysine, or serine) and screened these mutants for their biological activities in the presence and absence of estradiol. Two of the ER mutants, Y537A and Y537S, displayed estrogen-independent constitutive activity that was approximately 20% or 100%, respectively, of the activity of the wild type receptor with estradiol, when assessed in two different cell backgrounds using three different estrogen-responsive promoters. In some circumstances, the Y537E and Y537K proteins also exhibited some low level of constitutive activity. The constitutive activity of the mutants, as well as their activity in the presence of E2, was fully suppressed by antiestrogen. The extent of interaction of the constitutively active ERs with the steroid receptor coactivator-1 (SRC-1) closely parallel the magnitude of transcriptional activity of the receptor. Whereas wild type ER showed interaction with SRC-1 only in the presence of estrogen, Y537A and Y537S ER showed moderate or full interaction in the absence of ligand, an interaction that was blocked by antiestrogen, and the magnitude of interaction was increased to or remained at 100% upon estradiol treatment, implying that the ability of an ER to associate with SRC-1 is a good indicator of a transcriptionally active conformational state of the receptor. Our findings indicate that tyrosine 537 is in a region important in the ligand regulation of ER transcriptional activity and that the presence of certain amino acids at this position can shift ER into a conformation that is active even without ligand. However, tyrosine is not required at this site for estrogen binding or transcriptional response to estrogen in the systems investigated. Our findings, interpreted in light of the recently published x-ray crystal structure of the ligand-binding domains of three related receptors of the nuclear receptor superfamily, suggest that some of the amino acid substitutions introduced at position 537 may facilitate the shift of helix 12 of the ER into an active conformation and/or allow for differential stabilization of the receptor in its active form.