A homogenous polyphosphatase preparation was obtained from Saccharomyces cerevisiae cytosol with a 3.8% yield and 3540-fold purification. The enzyme hydrolysed polyphosphate (polyP) with various chain lengths, including polyP3, and split Pi off the end of the chain. It was inactive with respect to ATP, PPi, and p-nitrophenylphosphate. Its specific activity with polyP15 was 283 U/mg protein. The polyphosphatase was a monomeric protein with a molecular mass of 40 kDa. This enzyme was inactive without divalent cations and with Cu2+ and Ca2+. The ability of other divalent cations to activate the enzyme decreased in the following order; Co2+ > Mn2+ > Mg2+ > Zn2+. A kinetic model of the hydrolysis of polyP3 and action of Mg2+ is proposed.