In rodent uterus, both up- and down-regulation of estrogen receptor alpha (ERα) messenger ribonucleic acid (mRNA) and protein levels by estradiol has been demonstrated; however, it is not known which of the uterine compartments (endometrial epithelium, stroma, myometrium) respond to estradiol with autoregulation of ERα. The purpose of the present study was to investigate and compare the kinetics and cell type-specific effects of estradiol on uterine ERα expression in immature and adult rats. Ovariectomized female rats were injected s.c. with sesame oil or estradiol-17β. Uteri were collected and analyzed for changes in ERα mRNA using RNase protection assays (RPA) and in situ hybridization using radiolabeled probes specific for ERα. Immunohistochemical analysis was performed with a polyclonal antibody specific to ERα. Expression of ERα in the uterine epithelial cells decreased at 3 and 6 h after estradiol administration to immature and adult rats, respectively. At 24 h, ERα mRNA levels in the immature and mature rat uterus were higher than pretreatment levels but returned to baseline by 72 h. Pretreatment with cycloheximide did not block the 3-h repressive effect of estradiol, suggesting that the estradiol-induced decrease in ERα mRNA occurs independent of new protein synthesis. A decrease in ERα mRNA and protein was also observed in uterine epithelia at 3 and 6 h after an estradiol injection to immature and adult rats, and intensity of both the in situ hybridization signal and the immunostaining in the epithelium increased at 24 and 72 h. However, the periluminal stromal cells in the adult uterus and the majority of stromal cells of the immature uterus appeared to have increased ERα expression. The results indicate that down-regulation of ERα in the epithelia and up-regulation of stromal ERα play a role in early events associated with estradiol-induced cell proliferation of the uterine epithelia.