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Activation of hormone receptors was recently found to evoke oscillations of the cAMP concentration ([cAMP]) beneath the plasma membrane of insulin-secreting cells. Here we investigate how different time courses of cAMP signals influence the generation of cytoplasmic Ca2+ signals and nuclear translocation of the PKA (protein kinase A) catalytic subunit in individual INS-1 β-cells. [cAMP] was measured with a fluorescent translocation biosensor and ratiometric evanescent wave microscopy. Analysis of PKA nuclear translocation was performed with epifluorescence microscopy and FlAsH (fluorescein arsenical helix binder) labelling of tetracysteine-tagged PKA-Cα subunit. Both oscillatory and stable elevations of [cAMP] induced by intermittent or constant inhibition of phosphodiesterases with isobutylmethylxanthine evoked Ca2+ spiking. During [cAMP] oscillations, the Ca2+ spiking was restricted to the periods of elevated [cAMP]. In contrast, only stable [cAMP] elevation induced nuclear entry of FlAsH-labelled PKA-Cα. These results indicate that oscillations of [cAMP] lead to selective target activation by restricting the spatial redistribution of PKA. Abbreviations: CFP, cyan fluorescent protein; FlAsH, fluorescein arsenical helix binder; GLP-1, glucagon-like peptide-1; IBMX, isobutylmethylxanthine; PKA, protein kinase A; YFP, yellow fluorescent protein