Activated by thyroid hormone, the MAPK (ERK1/2) signaling pathway causes serine phosphorylation by MAPK of several nucleoproteins, including the nuclear thyroid hormone receptor β1. Because estrogen can activate MAPK and cause MAPK-dependent serine phosphorylation of nuclear estrogen receptor (ER)α, we studied whether thyroid hormone also promoted MAPK-mediated ERα phosphorylation. Human breast cancer (MCF-7) cells were incubated with physiological concentrations of l-T4 or 17β-estradiol (E2) for 15 min to 24 h, and nuclear ERα and serine-118-phosphorylated ERα were identified by Western blotting. Serine-118-phosphorylated ERα was recovered at 15 min in nuclei of MCF-7 cells exposed to either T4 or E2. The T4 effect was apparent at 15 min and peaked at 2 h, whereas the E2 effect was maximal at 4-6 h. T4-agarose was as effective as T4 in causing phosphorylation of ERα. T4 action on ERα was inhibited by PD 98059, an inhibitor of ERK1/2 phosphorylation, and by tetraiodothyroacetic acid, a T4 analog that blocks cell surface-initiated actions of T4 but is not itself an agonist. Electrophoretic mobility shift assay of nuclear extracts from T4-treated and E2-treated cells showed similar specific protein-DNA-binding. Indexed by [3H]thymidine incorporation and nuclear proliferating cell nuclear antigen, MCF-7 cell proliferation was stimulated by T4 and T4-agarose to an extent comparable with the effect of E2. This T4 effect was blocked by either PD 98059 or ICI 182,780, an ER antagonist. Thus, T4, like E2, causes phosphorylation by MAPK of nuclear ERα at serine-118 in MCF-7 cells and promotes cell proliferation through the ER by a MAPK-dependent pathway.