A distinct phosphodiesterasic activity (EC 3.1.4) was found in both mono- and dicotyledonous plants that catalyzes the hydrolytic breakdown of ADPglucose (ADPG) to produce equimolar amounts of glucose-1-phosphate and AMP. The enzyme responsible for this activity, referred to as ADPG pyrophosphatase (AGPPase), was purified over 1,100-fold from barley leaves and subjected to biochemical characterization. The calculated K(eq)' (modified equilibrium constant) value for the ADPG hydrolytic reaction at pH 7.0 and 25 degrees C is 110, and its standard-state free-energy change value (DeltaG') is -2.9 kcal/mol (1 kcal = 4.18 kJ). Kinetic analyses showed that, although AGPPase can hydrolyze several low-molecular weight phosphodiester bond-containing compounds, ADPG proved to be the best substrate (K(m) = 0.5 mM). P(i) and phosphorylated compounds such as 3-phosphoglycerate, PP(i), ATP, ADP, NADP(+), and AMP are inhibitors of AGPPase. Subcellular localization studies revealed that AGPPase is localized exclusively in the plastidial compartment of cultured cells of sycamore (Acer pseudoplatanus L.), whereas it occurs both inside and outside the plastid in barley endosperm. In this paper, evidence is presented that shows that AGPPase, whose activity declines concomitantly with the accumulation of starch during development of sink organs, competes with starch synthase (ADPG:1,4-alpha-d-glucan 4-alpha-d-glucosyltransferase; EC) for ADPG, thus markedly blocking the starch biosynthesis.