Successful blastocyst implantation requires extensive alteration of uterine epithelial cells (UECs) followed by their removal in order for the implanting blastocyst to penetrate into the underlying endometrium. The dynamics of focal adhesions (FAs) may play a role during this process because FAs are specialised structural complexes that provide adhesion between the cell and its underlying basal lamina. The present study investigated the expression and distribution of FA proteins in rat UECs at the time of implantation and their hormonal control as well as their expression in blastocyst stage embryos. Immunofluorescence microscopy showed that the principal focal adhesion proteins, talin and paxillin, were localised along the basal cell surface of UECs on day 1 of pregnancy and significantly reduced from the site of FAs at the time of implantation allowing the UECs to become less adhesive to the underling basal lamina. This is thought to be a critical process in the removal of UECs, which enables the invasion of the implanting blastocyst into the underlying decidual cells. Ovariectomised rats treated under various hormone regimes showed that talin and paxillin were tightly regulated by ovarian hormones in UECs. In particular, FA disassembly seen at the time implantation was predominantly under the control of progesterone. FA dynamics are regulated by the clustering of specific integrin subunits and immunofluorescence microscopy and immunoprecipitation have shown that integrinβ1 and β3 subunits colocalise and interact with talin at the site of FAs on day 1 of pregnancy. Integrin β1 and β3 were markedly reduced from the site of FAs at the time of implantation. However unlike the other FA proteins, localisation of integrin β3 on the apical membrane of UECs increased significantly at this time with a corresponding increase of integrin β3 expression in the membrane shown by subcellular fractionation. Like talin and paxillin, the distributional change of integrin β1 and β3 in the UECs at the time of implantation were also under the control of progesterone. Immunofluorescence microscopy have shown that in the rat blastocyst, integrin β3 was concentrated around the nuclei and was not present in the inner cell mass and integrin β1 weakly stained in the cytoplasm of the trophoblast cells. Interestingly, another FA protein, FAK was not present at the site of FAs in the UECs during early pregnancy. Instead, FAK colocalised with a tight junctional marker ZO-1 on day 1 of pregnancy and increased in the apical portion of the UECs at the time of implantation, suggesting a possible role of FAK in regulating tight junctional structures. FAK in the rat blastocyst was present in the apical membrane and did not colocalise with a tight junctional marker occludin, suggesting that FAK has a different role in the rat uterus and the blastocyst. Taken together, our results show that disassembly of FA proteins play an integral part in the removal of UECs in order to establish successful implantation and is tightly regulated by ovarian hormones. (platform)