Estrogen receptor (ER) is a ligand-inducible transcription factor that mediates the physiological effects of 17-estradiol (E2). In the uterus, E2 is involved in tissue growth, maintenance and differentiation. 5ER (5) is an ER variant protein expressed in uterine tumors but not in normal tissue. We examined the transcriptional activity of 5 and its modulation of human ER basal and E2-stimulated activity in Ishikawa cells, an endometrial cancer cell line. In transient transfection assays, 5 increased basal activity of an estrogen response element (ERE)-containing promoter in the absence or presence of ER, but lessened stimulation by ER and E2. Effects of 5 were not limited to model reporters, as cyclin D1 and complement 3 promoters were similarly affected. Increases in basal transcription required dimerization and DNA binding of 5, whereas decreased E2-stimulation with ER required only DNA binding. Decreased ligand stimulation was not unique to E2, but also applied to the selective estrogen receptor modulators tamoxifen and genistein. However, promoter stimulation by epidermal growth factor (EGF) is retained with 5. The ER coactivator small nuclear ring finger protein is expressed in Ishikawa cells and uterine tumors, and enhances effects of 5 alone and with ER on basal activity of an ERE reporter. Thus, in the presence of 5 plus ER there is a lower transcriptional response to E2 and SERMS, but stimulation by EGF is retained. The expression of 5 in uterine carcinoma may provide a mechanism by which tumors could maintain expression of E2-responsive genes in the absence of E2.