Inactivation of the PPN1 gene, encoding one of the enzymes involved in polyphosphate metabolism in the yeast Saccharomyces cerevisiae, was found to decrease exopolyphosphatase activity in the cytosol and vacuoles. This effect was more pronounced in the stationary growth phase than in the phase of active growth. The gene inactivation resulted in elimination of a approximately 440-kDa exopolyphosphatase in the vacuoles but did not influence a previously unknown vacuolar exopolyphosphatase with a molecular mass of >1000 kDa, which differed from the former enzyme in the requirement for bivalent cations and sensitivity to heparin. Inactivation of the PPN1 gene did not influence the level of polyphosphates in the cytosol but increased it more than twofold in the vacuoles. In this case, the polyphosphate chain length in the cytosol increased from 10-15 to 130 phosphate residues both in the stationary and active growth phases. In the vacuoles, the polyphosphate length increased only in the stationary growth phase. A conclusion can be made that the PPN1 gene product has different effects on polyphosphate metabolism in the cytosol and the vacuoles.