Saccharomyces cerevisiae mitochondria have a polyphosphatase activity which is insensitive to a number of inhibitors of mitochondrial ATPase and pyrophosphatase (PPase). Heparin (20 micrograms/ml) and EDTA (0.5 mM) do not inhibit ATPase and PPase activities but completely suppressed mitochondrial polyphosphatase activity. The mitochondrial polyphosphatase activity is maximal at neutral pH; it is inhibited by monovalent cations in the presence of Tris+ (K+ > Na+ > NH4+), and stimulated by bivalent metal cations (Co2+ > Mg2+ > Zn2+ > Mn2+). The polyphosphatase activity does not significantly depend on polyphosphate chain length from 9 to 208 but is more than one order of magnitude higher than activity with tripolyphosphate. Some properties of mitochondrial polyphosphatase activity differ from the characteristics of polyphosphatases of cell envelope, cytosol, vacuoles and nuclei of the same S. cerevisiae strain.