Saccharomyces cerevisiae nuclei possess a polyphosphatase activity which is insensitive to a number of inhibitors of ATPase and pyrophosphatase (PPase) activities of the same organelle. Heparin, an effective inhibitor of the nuclear polyphosphatase activity, does not alter either the ATPase and PPase activity. The nuclear polyphosphatase activity is optimal at pH 7.5. Bivalent metal cations stimulate this activity in the following order: Co2+ > Mg2+ > Zn2+ > Mn2+. However, the magnitude of the stimulating effect is much lower than that for the polyphosphatase activities from other organelles of the same yeast. The polyphosphatase activity is nearly the same for polyphosphates ranging from [symbol: see text] = 9 to [symbol: see text] = 208, but is 1.5 times higher for tripolyphosphate. The K(m) values for the hydrolysis of polyphosphates with chain lengths [symbol: see text] = 3, 15 and 208 are 100, 5 and 4.1 microM, respectively. The polyphosphatase activity differs in some properties from that of the cell envelope, cytosol and vacuoles of the same S. cerevisiae strain.