Accumulating evidence indicates that estrogen regulates diverse but interdependent signaling pathways via estrogen receptor (ER)-dependent and -independent mechanisms. However, the molecular relationship between these pathways for gene regulation under the direction of estrogen remains unknown. To address this possibility, our uterine analysis of Wnt/-catenin downstream effectors revealed that Lef-1 and Tcf-3 are up-regulated temporally by E₂ in an ER-independent manner. Lef-1 is abundantly up-regulated early (within 2 h), while Tcf-3 is predominantly induced after 6 h, and both sustained through 24 h. Interestingly, activated Lef-1/Tcf-3 molecularly interacted with ER in a time-dependent manner, suggesting they possess a cross-talk in the uterus by E₂. Moreover, dual immunofluorescence studies confirm their colocalization in uterine epithelial cells after E₂. Most importantly, using chromatin immunoprecipitation followed by PCR analyses, we provide evidence for an interesting possibility that ER and Tcf-3/Lef-1 complex occupies at certain DNA regions of estrogen-responsive endogenous gene promoters in the mouse uterus. By selective perturbation of activated Lef-1/Tcf-3 or ER signaling events, here we provide a novel evidence that cooperative interactions, by these two different classes of transcription factors at the level of chromatin, direct uterine regulation of estrogen-responsive genes. Collectively, these studies support a mechanism that integration of a nonclassically induced -catenin/Lef-1/Tcf-3 signaling with ER is necessary for estrogen-dependent endogenous gene regulation in uterine biology.