In breast cancer, epidermal growth factor (EGF) receptor (EGFR) expression is inversely correlated with expression of estrogen receptor (ER) and predicts the prognosis and failure of endocrine therapy. We report here, for the first time, that in ER-positive breast cancer cell lines, MCF-7, T47D, and BT474, 17β-estradiol (E2) transiently induced EGFR messenger RNA (mRNA) levels 2- to 3-fold; this induction was prevented by the presence of the antiestrogen ICI 164,384 and was also reflected in the level of EGFR protein. Up-regulation of EGFR mRNA is most likely due to a direct effect of ER on the EGFR gene, with no involvement of protein synthesis, as it was not inhibited in the presence of cycloheximide; however, the subsequent down-regulation of EGFR required de novo protein synthesis. E2 had no effect on EGFR mRNA stability, and EGFR transcript levels were found to parallel EGFR mRNA levels, further supporting a direct transcriptional mechanism in the regulation of EGFR expression by estrogens. Additionally, sequencing of the EGFR promoter revealed putative imperfect estrogen-responsive elements that were capable of binding human ER. The transient nature of EGFR induction by E2, with a rapid return to a basal level that is dependent on protein synthesis, suggests that breast cancer cells possess active mechanisms to maintain low levels of EGFR expression in the presence of estrogen and a functional ER.