The generation of cAMP is initiated when a ligand binds to a G protein-coupled receptor, stimulating the enzyme adenylyl cyclase to catalyze the cyclization of ATP. The production of cAMP within the cell is tightly regulated, in part through activities of cytoplasmic phosphodiesterases. The intracellular signaling of cAMP is coordinated primarily through two effector molecules: protein kinase A and exchange proteins directly activated by cAMP. Previous work has shown that PKA and Epac can have distinct, redundant, or even opposing effects within the same cell, and both play important roles in modulating host defense functions in macrophages. cAMP serves as a negative regulator of phagocyte function and elevated cAMP levels are associated with suppression of innate immune functions including the production of proinflammatory mediators, phagocytosis, and microbial killing. Early biochemical and fixed cell microscopy studies Sanggenone-D indicated that intracellular cAMP production in macrophages and neutrophils increases during phagocytosis, through regulation by PDEs. More recent work has shown that PDEs play an important role in creating discrete subcellular pools of cAMP within the cell, with higher levels of cAMP found at the plasma membrane and within the nucleus and lower levels in the cytosol. Studies employing classical biochemical and fixed-cell microscopy approaches obtain suboptimal kinetic and spatial resolution of cAMP pools. In recent years, the use of techniques based on Fo��rster resonance energy transfer have allowed monitoring of cAMP levels in live cells. This provides better spatial and kinetic information about intracellular cAMP dynamics. FRET microscopy has demonstrated that cAMP compartmentalization plays an important role in mediating intracellular signaling events. A wide range of cAMP biosensors have been utilized, and these differ considerably in their localization, dynamic range, temporal resolution and signal-tonoise ratios.This study investigated the spatial and temporal dynamics of cAMP in live phagocytosing macrophages. Using cAMP FRET biosensors, we show that levels of cAMP rise quickly at the nascent Diatrizoic acid phagocytic cup and return to baseline following internalization of the particle.