The identification and characterization of estrogen regulated genes in reproductive tissues is an important step in understanding estrogen's mechanism of action in sexual development and neoplasia. It is also important, given the clinical interest, to evaluate the molecular effects of estrogen agonists/antagonists such as tamoxifen and droloxifene in reproductive tissues. In this report, our goal was to identify estrogen regulated genes in the uterus and to compare the regulation by estrogen and tamoxifen with that of droloxifene. A subtractive cDNA library strategy was developed to identify estrogen-regulated genes in the uteri of ovariectomized rats 4 h after treatment with 17-alpha-ethynyl estradiol (30 microg/kg). The mRNAs encoding 8 genes were confirmed by Northern blot analysis to be induced at early times following estrogen administration. Calcium binding protein 9 kDa and complement protein 3 are well characterized estrogen regulated genes that were identified in the library and served as markers for estrogen action. In addition, mRNAs encoding the interleukin 4 receptor, heat-shock protein 70 kDa, metallothionein, tumor necrosis factor regulated gene 6, inositol-1-monophosphate synthase, and cyr-61 were induced in the uterus by estrogen. The identified mRNAs were then examined for regulation by droloxifene (1 and 10 mg/kg, p.o.) and tamoxifen (10 mg/kg, p.o.). Both droloxifene and tamoxifen induced mRNA levels for all of these genes. However, clear quantitative and temporal differences were observed when comparing estrogen versus droloxifene versus tamoxifen. For example, estrogen induced IL4 receptor mRNA to a greater degree than did tamoxifen or droloxifene. Conversely, tamoxifen resulted in a much greater induction of cyr61 than did either estrogen or droloxifene. Droloxifene at 1 mg/kg, an efficacious dose for prevention of bone loss in this model, did not or only slightly induced the mRNA for all of the genes examined with the exception of cyr61. In conclusion, the modified subtractive library method used in this study proved to be efficient in the identification of estrogen-regulated genes in the uterus. The identities of the regulated genes were consistent with the concept that estrogen functions to prime uterine tissue for increased responsivity to extracellular signals such as growth factors and cytokines. Elucidating the physiological role of these newly identified estrogen responsive genes and the mechanisms responsible for the different responses to droloxifene versus estrogen and tamoxifen may be important in enhancing our understanding of tissue selective estrogen agonists/antagonists.