Phosphorylation of Ser118 of human estrogen receptor alpha (ER) enhances ER-mediated transcription and is induced by hormone binding and by activation of the mitogen-activated protein kinase (MAPK) pathway. We discovered that phosphorylation of Ser118 reduces the electrophoretic mobility of the ER. Using this mobility shift as an assay, we determined the in vivo stoichiometry and kinetics of Ser118 phosphorylation in response to estradiol, ICI 182,780, epidermal growth factor (EGF), and phorbol 12-myristate 13-acetate (PMA). In human breast cancer MCF-7 cells, estradiol induced a steady state phosphorylation of Ser118 within 20 min with a stoichiometry of 0.67 mol of phosphate/mol of ER. Estradiol did not activate p42/p44 MAPK, and basal p42/p44 MAPK activity was not sufficient to account for phosphorylation of Ser118 in response to estradiol. In contrast, both EGF and PMA induced a rapid, transient phosphorylation of Ser118 with a stoichiometry of approximately 0. 25, and the onset of Ser118 phosphorylation correlated with the onset of p42/p44 MAPK activation by these agents. Either the EGF- or PMA-induced Ser118 phosphorylation could be inhibited without influencing estradiol-induced Ser118 phosphorylation. The data suggest that a kinase other than p42/p44 MAPK is involved in the estradiol-induced Ser118 phosphorylation. We propose that the hormone-induced change in ER conformation exposes Ser118 for phosphorylation by a constitutively active kinase.