The estrogen receptor (ER) appears to be down-regulated by its own ligand in some estrogen (E2)-responsive tissues as well as in cell lines such as MCF-7 and GH3. Surprisingly, we observed ER down-regulation in a newly constructed E2-responsive cell line (Rat1 + ER), in which expression of the coding region of the ER cDNA was driven by the Moloney murine leukemia virus long terminal repeat. We present evidence that the coding region of the ER cDNA, but not the Moloney murine leukemia virus long terminal repeat, possesses a sequence(s) necessary for ER down-regulation. The observed down-regulation occurs at ligand-binding and protein, as well as mRNA, levels. Marked decreases in both protein and mRNA levels were observed as early as 3 h after E2 treatment. Furthermore, maximal down-regulation occurred by 18-24 h with ligand-binding, and mRNA levels reached approximately 20% that of controls. ER down-regulation in Rat1 + ER cells is only partially inhibited by the presence of cycloheximide and therefore suggests a direct participation of the ER in this process. E2 does not appear to influence the stability of the ER transcript, which implies that negative regulation is occurring at the transcriptional level. Finally, since we can demonstrate ER binding to a portion of the cDNA sequence, we propose a mechanism whereby ER binding to its putative negative element leads to transcriptional repression of the upstream promoter.