Regulation of progesterone receptor (PR) in uterine stroma (endometrial stroma plus myometrium) by estrogen was investigated in estrogen receptor-α (ERα) knockout (αERKO) mice. 17β-Estradiol (E₂) increased PR levels in uterine stroma of ovariectomized αERKO mice, and ICI 182 780 (ICI) inhibited this E₂-induced PR expression. Estrogen receptor-β (ERβ) was detected in both uterine epithelium and stroma of wild-type and αERKO mice by immunohistochemistry. In organ cultures of αERKO uterus, both E₂ and diethylstilbestrol induced stromal PR, and ICI inhibited this induction. These findings suggest that estrogen induces stromal PR via ERβ in αERKO uterus. However, this process is not mediated exclusively by ERβ, because in ERβ knockout mice, which express ERα, PR was up-regulated by E₂ in uterine stroma. In both wild-type and αERKO mice, progesterone and mechanical traumatization were essential and sufficient to induce decidual cells, even though E₂ and ERα were also required for increase in uterine weight. Progesterone receptor was strongly expressed in decidual cells in αERKO mice, and ICI did not inhibit decidualization or PR expression. This study suggests that up-regulation of PR in endometrial stroma is mediated through at least three mechanisms: 1) classical estrogen signaling through ERα, 2) estrogen signaling through ERβ, and 3) as a result of mechanical stimulation plus progesterone, which induces stromal cells to differentiate into decidual cells. Each of these pathways can function independently of the others.