The existence of a specific inorganic pyrophosphatase (PPi ase), distinct from alkaline phosphatase in bone has been suggested but is often regarded as questionable. In the present investigation, several features of PPi ase activity have been demonstrated, which suggest that it represents an enzyme protein different from those splitting phosphomonoesters and ATP. PPi ase was largely destroyed during extraction with n-butanol, which facilitated the solubilization of alkaline phosphatase and ATP splitting enzymes and only partially destroyed acid phosphatase. Two major groups of phosphate esters and pyrophosphates splitting enzymes were separated by gel filtration from homogenates of rat bones. The first pool contained high ATP-ase and phosphomonoesterase activities, but only low activity against inorganic pyrophosphate (PPi) in the presence of MgCl2. The second pool was most active against PPi at pH 7.5 in the presence of excess MgCl2 and only slightly hydrolyzed phosphomonoesters or ATP. Immunodiffusion showed that these 2 pools contained 2 distinct proteins. It was concluded that there exists a specific inorganic pyrophosphatase distinct from phosphomonoesterases and ATP-ases in bone tissue.