Estrogens are thought to promote labor by increasing the expression of pro-contraction genes in myometrial cells. The specific estrogen receptors ((ERs: ERα and ERβ (also known as ESR1 and ESR2)) and G protein-coupled receptor 30 (GPR30; also known as G protein-coupled estrogen receptor 1)) and signaling pathways that mediate these actions are not clearly understood. In this study, we identified the ERs expressed in the pregnant human myometrium and determined a key extranuclear signaling pathway through which estradiol (E(2)) modulates expression of the gene encoding the oxytocin receptor (OXTR), a major pro-contraction protein. Using quantitative RT-PCR, we found that ERα and GPR30 mRNAs were expressed in the human pregnant myometrium while ERβ mRNA was virtually undetectable. While mRNA encoding ERα was the predominant ER transcript in the pregnant myometrium, ERα protein was largely undetectable in myometrial tissue by immunoblotting. Pharmacological inhibition of 26S proteasome activity increased ERα protein abundance to detectable levels in term myometrial explants, however, indicating rapid turnover of ERα protein by proteasomal processing in the pregnant myometrium. E(2) stimulated rapid extranuclear signaling in myometrial explants, as evidenced by increased extracellularly regulated kinase (ERK1/2) phosphorylation within 10 min. This effect was inhibited by pre-treatment with an ER antagonist, ICI 182 780, indicating the involvement of ERα. Inhibition of ERK signaling abrogated the ability of E(2) to stimulate OXTR gene expression in myometrial explants. We conclude that estrogenic actions in the human myometrium during pregnancy, including the stimulation of contraction-associated gene expression, can be mediated by extranuclear signaling through ERα via activation of the ERK/mitogen-activated protein kinase pathway.