Transformation of the endometrium into the receptive phase is under the control of ovarian steroid hormones and is modulated by embryonic signals during implantation. We have previously shown that this differentiation process is accompanied by a suppression of gap junction connexins (Cx) 26 and 43 before implantation followed by a local induction of both connexins in the implantation chamber. In the present study, we demonstrate that connexin gene expression in the rodent endometrium is regulated via two distinct signaling pathways during these different stages of early pregnancy. During preimplantation, transcription of connexins can be induced by estrogen via an estrogen receptor (ER)-dependent pathway. Additionally, Cx26 and Cx43 are induced by embryonic signals during implantation and delayed implantation as well as during artificially induced decidualization. In contrast to the estrogen-induced expression, this embryonic/decidual-associated induction of Cx26 and Cx43 could not be blocked by antiestrogen, thus pointing to another regulatory pathway independent of the ER. Studies in ERalpha and ERbeta knockout mice confirmed these different pathways, demonstrating that in the endometrium, estrogen-mediated Cx26 gene induction, but not induction during decidualization, is dependent on functional ERalpha. To evaluate potential embryonic signals regulating Cx26 expression, uteri of pseudopregnant animals were incubated with different mediators in an organ-culture model, showing that catechol estrogen and mediators of the inflammatory cascade such as prostaglandin F(2alpha) and interleukin-1beta are able to induce Cx26 expression through the ER-independent pathway. Thus, the present study demonstrates that endometrial expression of Cx26 and Cx43 is induced via estrogen and ERalpha during preimplantation but then utilizes an ER-independent signaling pathway during embryo implantation and decidualization.