Epac belongs to a new family of proteins that can directly mediate the action of the intracellular second messenger cAMP by activating a downstream small GTPase Rap1. The Epac/Rap1 pathway represents a novel cAMP-signaling cascade that is independent of the cAMP-dependent protein kinase (PKA). In this study, we have used fluorescence microscopy to probe the intracellular targeting of Epac during different stages of the cell division cycle and the structural features that are important for Epac localization. Our results suggest Epac, endogenous or expressed as a green fluorescent protein fusion protein, is mainly localized to the nuclear membrane and mitochondria during interphase in COS-7 cells. Deletion mutagenesis analysis reveals that whereas the DEP domain is responsible for membrane association, the mitochondrial-targeting sequence is located at the N terminus. Although Epac predominantly exhibits perinuclear localization in interphase, the subcellular localization of Epac is cell cycle-dependent. Epac disassociates from the nuclear membrane and localizes to the mitotic spindle and centrosomes in metaphase. At the end of the cell cycle, Epac is observed to reassociate with the nuclear envelope and concentrate around the contractile ring. Furthermore, overexpression of Epac in COS-7 cells leads to an increase in multinuclear cell populations. These results suggest that Epac may play an important role in mitosis.