An inorganic pyrophosphatase [E.C. 3.6.1.1] was isolated from Methanothrix soehngenii. In three steps the enzyme was purified 400-fold to apparent homogeneity. The molecular mass estimated by gelfiltration was 139 +/- 7 kDa. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis indicated that the enzyme is composed of subunits with molecular masses of 35 and 33 kDa in an alpha 2 beta 2 oligomeric structure. The enzyme catalyzed the hydrolysis of inorganic pyrophosphate, tri- and tetrapolyphosphate, but no activity was observed with a variety of other phosphate esters. The cation Mg2+ was required for activity. The pH optimum was 8 at 1 mM PPi and 5 mM Mg2+. The enzyme was heat-stable, insensitive to molecular oxygen and not inhibited by fluoride. Analysis of the kinetic properties revealed an apparent Km for PPi of 0.1 mM in the presence of 5 mM Mg2+. The Vmax was 590 mumol of pyrophosphate hydrolyzed per min per mg protein, which corresponds to a Kcat of 1400 per second. The enzyme was found in the soluble enzyme fraction after ultracentrifugation, when cells were disrupted by French Press. Upto 5% of the pyrophosphatase was associated with the membrane fraction, when gentle lysis procedure were applied.