Cyclic adenosine 3', 5'- monophosphate (cAMP) is a second messenger that functions through binding to its downstream targets protein kinase A (PKA), cyclic-regulated ion channels (CNG channels) and Epac1 (exchange protein directly activated by cAMP). Epac1 is guanine nucleotide exchange factor toward both Rap1 and Rap2. It is kept in the inactive conformation by an intramolecular interaction between the regulatory and catalytic domain. Binding of cAMP to the cAMP binding domain liberates the catalytic domain, resulting in the activation of Epac. In order to visualize the conformational change between inactivate- and active- state of Epac, a CFP-Epac-YFP probe was generated and fluorescence resonance energy transfer (FRET) between the two fluorescent moieties was monitored in vivo. The FRET signal was rapidly decreased in response to cAMP-raising agents, whereas it fully recovers after addition of cAMP-lowering agonists. This indicates that cAMP causes a significant conformational change in vivo and supports the unfolding model for Epac1 activation. To unravel more functions of Epac1, a series of Epac1 antibodies (Abs) were generated and characterized. 5D3, one of Epac1 monoclonal Abs, was further characterized based on its ability to recognize activate conformation Epac1. In vitro, the epitope of 5D3 was mapped within the cAMP binding domain, especially around Leucine 273. This region is hidden during autoinhibition. Using Epac1 Abs, subcellular localization of Epac1 was investigated in detail. Epac1 is mainly distributed around perinuclear region, especially in the endoplasmatic reticulum and the Golgi apparatus, as well as in the plasma membrane, especially at microvilli in fully polarized cells. Functional domains which are responsible for proper localization of Epac1 were also analyzed in detail. Our results revealed that both DEP domain and the EzB domain (the first 49 aa) are required for correct localization of Epac1 and the activation of Rap. The EzB domain is not only responsible but also sufficient for targeting Epac1 at microvilli. In contrast the DEP domain is only responsible for membrane localisation. Importantly, the microvillar localization is through binding to Ezrin/Radixin, proteins that function as linkers between the actin cytoskeleton and the apical membrane of polarized cells and as scaffold protein for protein complexes. The functional relevance of this interaction is still unclear, but the mere fact that Epac1 only binds to the active form of Ezrin/Radixin indicates that activation of these proteins is a crucial step in the spatial regulation of Epac1. Nuclear accumulation of Epac was also observed after removing the EzB domain. This effect could be mimicked by stimulation of the cells with HGF, that induces cell scattering, and with overexpression of RapV12 that induces cell spreading. These results suggest that upon loss of cell polarity and thus disruption of the apical structures, part of Epac1 translocation to the nucleus. However, the function of Epac1 in the nucleus remains a question mark.