Pyrophosphatase, which splits specifically reduced pyridine nucleotide (NADH_2,NADPH_2) into reduced nicotineamide mononucleotide and AMP or 3', 5'-ADP, has been purified 80 times from rat liver acetone powder. The purified enzyme indicated the turnover rate of 77 μmoles/hr/mg protein and splits only reduced pyridine nucleotide, but not splits oxidized NAD or NADP. Pyrophosphatases were found to be tightly bound to particle fraction. Intracellurar distribution was studied and mitochondrial and microsomal pyrophosphatases were separated clealy on DEAE-cellulose column chromatography, but kinetic difference between mitochondrial and microsomal isozymes were not recognized. The considerable degree of inhibition was recognized by addition of oxidized NAD, NADP, adeninenucleotides or reduced nicotineamide mononucleotide.