Objective: Our goal was to demonstrate expression and functionality of oxytocin receptors in a human endometrial cell line. This cell line could then be used for further investigation of the role of oxytocin in reproductive function at the cellular level. Study Design: Oxytocin receptor messenger ribonucleic acid expression was determined by reverse transcriptase–polymerase chain reaction deoxyribonucleic acid amplification with ribonucleic acid from confluent Ishikawa cells. Ligand binding to whole cells was evaluated by nonlinear regression analysis with an iodinated oxytocin antagonist. The coupling of the oxytocin receptor to signaling pathways was evaluated by measuring oxytocin-stimulated increases in intracellular calcium concentration, phosphorylation of ERK2 (extracellular-regulated protein kinase 2) mitogen-activated protein kinase, and prostaglandin E₂ release. Results: Polyacrylamide gel electrophoresis of the reverse transcriptase–polymerase chain reaction products demonstrated the presence of oxytocin receptor messenger ribonucleic acid in Ishikawa cells. Ligand-binding analysis of these cells demonstrated a single class of noninteracting sites, with a B_max (maximal number of binding sites) of 77.7 fmol/mg deoxyribonucleic acid and an apparent dissociation constant of 8.3 × 10^–11 mol/L. Stimulation with 100-nmol/L oxytocin caused a rapid transient increase in intracellular free calcium concentration, which was blocked by 1-μmol/L oxytocin antagonist. Treatment of cells with oxytocin for 10 minutes resulted in a marked increase in the phosphorylation of ERK2, as determined by Western blot analysis, and a 5-fold increase in prostaglandin E₂ release. Conclusion: This study is the first to demonstrate functional oxytocin receptors in an established human endometrial cell line. This cell line will be useful in elucidating the mechanisms of action of oxytocin in the reproductive tract at the molecular level. (Am J Obstet Gynecol 2000;182:850-5.)