We studied physiological roles of the yeast vacuole in the phosphate metabolism using 31P-in vivo nuclear magnetic resonance (NMR) spectroscopy. Under phosphate starvation wild-type yeast cells continued to grow for two to three generations, implying that wild-type cells contain large phosphate pool to sustain the growth. During the first four hours under the phosphate starved condition, the cytosolic phosphate level was maintained almost constant, while the vacuolar pool of phosphate decreased significantly. 31P-NMR spectroscopy on the intact cells and perchloric acid (PCA) extracts showed that drastic decrease of polyphosphate took place during this phase. In contrast, Δslp1 cells, which were defective in the vacuolar compartment, thus lacked polyphosphate, ceased their growth immediately when they faced to phosphate starvation. Taken together, we conclude that vacuolar polyphosphate provides an active pool for phosphate and is mobilized to cytosol during phosphate starvation and sustained cell growth for a couple rounds of cell cycle.