Here, we studied the potential role of inorganic polyphosphate (polyP) as an energy source for ADP and ATP formation in the extracellular space. In SaOS-2 cells, we show that matrix vesicles are released into the extracellular space after incubation with polyP. These vesicles contain both alkaline phosphatase (ALP) and adenylate kinase (AK) activities (mediated by ALPL and AK1 enzymes). Both enzymes translocate to the cell membrane in response to polyP. To distinguish the process(es) of AMP and ADP formation during ALP hydrolysis from the ATP generated via the AK reaction, inhibition studies with the AK inhibitor A(5')P5(5')A were performed. We found that ADP formation in the extracellular space occurs after enzymatic ATP synthesis. After exposure to polyP, a significant increase of the ADP level was observed, which is likely to be been catalyzed by ALP. This increase is not due to an intensified ATP release via exocytosis. The ATP level in the extracellular space of SaOS-2 cells is strongly increased in response to polyP, very likely mediated by the AK. We propose that the ALP and AK enzymes are involved in the extracellular ADP and ATP synthesis.