Rat-liver nuclei contain a soluble inorganic pyrophosphatase. This enzyme differs from other animal pyro-phosphatases which are partially associated with glucose-6-phosphatase activity in the following properties: unusually high substrate affinity, pH-optimum, solubility, thermal instability, and intracellular dis-tribution. The maximal rate of pyrophosphate hydrolysis at pH 7.3 is 0.4 [mu] moles per min per mg soluble nuclear protein. Magnesium pyrophosphate has been found as the actual substrate (Km=6.6xl0-6 [image]); in addition, Mg2 ions are essential for the activity (Km = 3.2x1O-4 [image]). The substrate and the activating Mg2 act at 2 different functional sites on the enzyme. Free pyrophosphate competitively inhibits at the substrate site (Ki about 2x1O-7 [image]), whereas higher complexes of mag-nesuim pyrophosphate seem to exert a competitive inhibition at activator site (Ki = 1 x 10-6 [image]). The enzyme demonstrates a marked substrate-specificity for inorganic pyrophosphate; condensed poly-and metaphosphates are split to the maximum extent of 1% of the rate of pyrophosphate hydrolysis, nucleoside phosphates are not cleaved at all. The specificity for the activating metal ion is also very high. No other divalent cation which was studied, could replace Mg. All the metal ions tested lead to an inhibition of the enzyme-Ca29 being the most potent-probably by competition with Mg2 for the activator site. NAD and ATP are strong inhibitors of the nuclear inorganic pyro-phosphatase. NAD acts in a competitive manner at the substrate site (Ki = 5.7x 1O-5 [image]), ATP, however, acts in a competitive manner at the activator site (Ki = 2.5x1O-4 [image]), probably by chelation with the free Mg2 ions. The metabolic function of the enzyme is most probably that it allows the NAD-synthesis in that the equilibrium of the NAD-phosphorylase reaction is reversed, which otherwise would oppose the NAD-synthesis. Influences of metabolites (NAD, ATP) and magnesium lead to multiple possibilities of controlling the rate of enzymatic pyrophosphate hydrolysis in rat-liver nuclei.