Objective: To test the hypothesis that an abnormality in glycogen synthase kinase-3 (GSK3) is a pathogenic factor in polycystic ovary syndrome (PCOS). Design: Prospective experimental study (adipocytes). Setting: Tertiary-care academic medical center and teaching hospital. Patient(s): Twenty patients with PCOS and 21 healthy control women. Intervention(s): Blood sampling, physical exam, biopsy of SC lower abdominal fat. Main Outcome Measure(s): Glucose transport and protein levels and phosphorylation state of glycogen synthase kinase (GSK)-3α and GSK3β in adipocytes; assessment of GSK3β activity. Result(s): Basal protein levels of glycogen synthase kinase (GSK3α and GSK3β) did not differ between control women and women with PCOS, nor did basal or insulin-stimulated levels of serine phosphorylated GSK3α. However, in adipocytes of women with PCOS, insulin stimulation was not associated with increased serine phosphorylation of GSK3β, in contrast to the case of control women. Tyrosine phosphorylation of GSK3β also was higher in women with PCOS, compared with in control women. Consistent with the phosphorylation data, GSK3β activity was elevated in PCOS adipocytes. Conclusion(s): These data suggest that GSK3β is hyperactivated and resistant to down-regulation by insulin in PCOS. By using physiologic approaches, we demonstrated that abnormal GSK3β regulation is a potential mechanism for the insulin resistance that is seen in some women with PCOS, which may contribute to their development of the syndrome. © 2008 American Society for Reproductive Medicine.