In mice, the uterus becomes transiently receptive to the hatched blastocyst on the day of implantation to allow its attachment to the luminal epithelium and subsequent invasion into the uterus. This uterine preparation for implantation is regulated by estradiol-17beta and progesterone, acting through their transcription factor receptors. Using ovariectomized mice treated with physiological regimens of these hormones, combined with methods to isolate RNA specifically from the uterine epithelium followed by transcriptome analysis on cDNA microarrays, 222 genes whose transcript abundance was specifically increased by estradiol-17beta and progesterone treatment were identified. Gene ontology analysis revealed an emphasis on genes involved with immune responses, extracellular matrix metabolism, and cell-to-cell communication. In situ hybridization to uterine sections isolated through the first 6 d of pregnancy identified novel sets of genes such as Bach, Myd88, Cd14, Isg20, and Lrp2 whose expression was restricted to the uterine epithelium during the implantation window. Particularly notable was the expression of the mRNA for members of the signaling pathway from the Toll-like receptors to its downstream targets such as Irg-1. The identification of these genes showing a cell type hormonally regulated pattern of expression in the uterus suggests novel functions for them during implantation.