Clofibrate increased inorganic pyrophosphatase (PPase) activity in peroxisomes more than 12-fold (740 milliunits/head, 15.9 +/- 5.0 milliunits/mg protein) in rat liver. The distribution of cytochrome c oxidase and that of the PPase in a Nycodenz gradient suggested that the PPase is an original peroxisomal enzyme and not a mitochondrial contaminant: This was confirmed by second Nycodenz gradient centrifugation. The optimum pH of the peroxisomal PPase was about 8.5. The activity was specific to inorganic pyrophosphate (PPi), the Km value for PPi being 34.1 +/- 3.3 microM. It was strictly dependent on Mg2+ and showed a sigmoidal dose-response for Mg2+ with an S0.5 value of 100 microM. The activity was inhibited by Ca2+, p-chloromercuriphenylsulfonic acid, Hg2+, N-ethylmaleimide, and NaF. The functions of peroxisomal PPase are discussed.