Estrogen is of great importance in the regulation of uterine function. The aim of this study was to examine the individual physiological roles of each of the two receptors for estradiol, estrogen receptor (ER) and estrogen receptor (ER), and their potential co-modulatory effects on gene expression and uterine growth using recently developed ER subtype-selective agonist ligands. The use of ER subtype-selective ligands provides an alternative, complementary approach to the use of receptor knockout mice. Administration of the ER selective ligand propyl pyrazole triol (PPT) to immature mice resulted in a significant increase in uterine weight, as well as BrdU incorporation and proliferating cell nuclear antigen (PCNA) expression in luminal epithelial cells. PPT also increased complement C3, lactoferrin and glucose-6-phosphate dehydrogenase (G6PDH), and decreased androgen receptor (AR) and progesterone receptor (PR) mRNA levels in uterine tissue, as did estradiol (E₂). However, when compared with E₂, PPT was less effective in stimulating uterine weight, complement C3 and G6PDH expression but was as effective as E₂ in regulating lactoferrin, AR, and PR expression. In contrast to the action of the ER agonist PPT, the ER agonist diarylpropionitrile (DPN) did not increase uterine weight or luminal epithelial cell proliferation at a dose that reduced G6PDH and elicited a decrease in PR and AR mRNA and protein expression. Interestingly, DPN reduced the uterine weight stimulation by PPT, and enhanced the effect of PPT in decreasing uterine PR and AR mRNA. These findings with ER subtype-selective ligands indicate that ER is the major regulator of estrogen function in the uterus, but that ER does exert effects on some uterine markers of estrogen action. In addition, ER can modulate ER activity in a response-specific and dose-dependent manner.