Escherichia coli and potato (Solanum tuberosum) ADP-sugar pyrophosphatases (EcASPP and StASPP, respectively) are 'Nudix' hydrolases of the bacterial glycogen and starch precursor molecule, ADP-glucose (ADPG). We have previously shown that potato leaves expressing EcASPP either in the cytosol or in the chloroplast exhibited large reductions in the levels of starch, suggesting the occurrence of cytosolic and plastidial pools of ADPG linked to starch biosynthesis. In this work, we produced and characterized potato and Arabidopsis plants expressing EcASPP and StASPP fused with green fluorescent protein (GFP). Confocal fluorescence microscopy analyses of these plants confirmed that EcASPP-GFP has a cytosolic localization, whereas StASPP-GFP occurs in the plastid stroma. Both source leaves and potato tubers from EcASPP-GFP-expressing plants showed a large reduction of the levels of both ADPG and starch. In contrast, StASPP-GFP-expressing leaves and tubers exhibited reduced starch and normal ADPG contents when compared with control plants. With the exception of starch synthase in StASPP-GFP-expressing plants, no pleiotropic changes in maximum catalytic activities of enzymes closely linked to starch metabolism could be detected in EcASPP-GFP- and StASPP-GFP-expressing plants. The overall data (i) show that potato plants possess a plastidial ASPP that has access to ADPG linked to starch biosynthesis and (ii) are consistent with the occurrence of plastidic and cytosolic pools of ADPG linked to starch biosynthesis.