Activated by thyroid hormone, the MAPK (ERK1/2) signaling pathway causes serine phosphorylation by MAPK of several nucleoproteins, including the nuclear thyroid hormone receptor beta1. Because estrogen can activate MAPK and cause MAPK-dependent serine phosphorylation of nuclear estrogen receptor (ER)alpha, we studied whether thyroid hormone also promoted MAPK-mediated ERalpha phosphorylation. Human breast cancer (MCF-7) cells were incubated with physiological concentrations of l-T(4) or 17beta-estradiol (E(2)) for 15 min to 24 h, and nuclear ERalpha and serine-118-phosphorylated ERalpha were identified by Western blotting. Serine-118-phosphorylated ERalpha was recovered at 15 min in nuclei of MCF-7 cells exposed to either T(4) or E(2). The T(4) effect was apparent at 15 min and peaked at 2 h, whereas the E(2) effect was maximal at 4-6 h. T(4)-agarose was as effective as T(4) in causing phosphorylation of ERalpha. T(4) action on ERalpha was inhibited by PD 98059, an inhibitor of ERK1/2 phosphorylation, and by tetraiodothyroacetic acid, a T(4) analog that blocks cell surface-initiated actions of T(4) but is not itself an agonist. Electrophoretic mobility shift assay of nuclear extracts from T(4)-treated and E(2)-treated cells showed similar specific protein-DNA-binding. Indexed by [(3)H]thymidine incorporation and nuclear proliferating cell nuclear antigen, MCF-7 cell proliferation was stimulated by T(4) and T(4)-agarose to an extent comparable with the effect of E(2). This T(4) effect was blocked by either PD 98059 or ICI 182,780, an ER antagonist. Thus, T(4), like E(2), causes phosphorylation by MAPK of nuclear ERalpha at serine-118 in MCF-7 cells and promotes cell proliferation through the ER by a MAPK-dependent pathway.