Progesterone pretreatment of ovariectomized rat uteri increases the number of synchronously proliferating stromal cells in response to estradiol 17-β. To identify the signals involved in stimulating synchronous proliferation, sexually mature ovariectomized rats were injected with progesterone (2 mg) for 3 consecutive days. Estradiol 17-β (0.2 μg) was administered to initiate cell cycle entry. Uterine samples were removed at various times after hormone administration and changes in wingless (Wnt) pathway effectors and gene targets were identified by microarray. Progesterone pretreatment decreased glycogen synthase kinase-3β (GSK-3β) and increased expression of T-cell factor/lymphoid enhancer factor (TCF/LEF). GSK-3β protein decreased markedly in the uterine stroma of progesterone-pretreated uteri with the concomitant appearance of β-catenin in these stromal cells. Translocation of β-catenin from the cytosol to the nuclei in progesterone-pretreated stromal cells was stimulated in response to estradiol. β-Catenin binding to TCF/LEF increased (P<0.05) in progesterone-pretreated uteri in response to estradiol. Progesterone stimulated the expression of the Wnt target gene urokinase plasminogen activator receptor (uPA-R) in the periluminal uterine stromal cells. The expression of uPA-R increased in progesterone-pretreated stromal cells in response to estradiol administration. Together, the results indicate that progesterone initiates Wnt signaling in the uterine stroma by down-regulating GSK-3β. However, nuclear translocation of β-catenin and sufficient complex formation with TCF/LEF to activate stromal cell cycle entry requires estradiol. Stimulation of a uterine stromal cell line to proliferate and differentiate resulted in β-catenin accumulation, suggesting that endocrine-dependent Wnt signaling controls proliferation and differentiation (decidualization).