In addition to the nonspecific alkaline and acid phosphatases, mammalian tissues and serum contain another phosphatase, designated as 5-nucleotidase.' 8 The 5-nucleotidase of bull semen has been purified and well characterized by Heppel and Hilmoe,3 and the enzyme present in human serum appears to be similar to it in many respects:Serum 5-nucleotidase can be distinguished from the nonspecific phosphatases by a number of characteristics, including :This enzyme specifically hydrolyzes nucleotides in which the phosphate is attached to the 5 position of the pentose, such as adenosine-5-phosphate and inosine-5-phosphate. Adenosine-3-phosphate and other phosphate esters, including P-glycerophosphate and phenyl-phosphate, are unaffected.(2) Optimum p H . Serum 5-nucleotidase is an alkaline phosphatase with a p H optimum between 7.5 and 8.0.(3) Effect of magnesium ion. Addition of magnesium ion to the substrate in optimum concentration results in a marked activation of 5-nucleotidase, averaging a threefold increase in activity.Serum 5-nucleotidase has a greater resistance to inactivation by EDTA than does the nonspecific alkaline phosphatase.The activity of 5-nucleotidase is consistently localized to the @globulin fraction of both normal and pathological human sera, whereas both the nonspecific acid and alkaline phosphatases are a-2-globulins.Assay of 5-nucleotidase in serum by determining the rate of hydrolysis of a 5-nucleotide such as adenosine-5-phosphate is complicated by the fact that the nonspecific alkaline phosphatase of serum also hydrolyzes this substrate. This has been circumvented by preliminary incubation of serum with 0.0015 M ethylenediaminetetraacetic acid, which inactivates the alkaline phosphatase, but not the 5-nucleotidase. The rate of hydrolysis of adenosine-5-phosphate has then been determined a t p H 7.5 in the presence of 0.04 M magnesium ion, and the unit of 5-nucleotidase activity has been designated as equivalent to the liberation of 1 mg. of phosphate ion in 1 hour; this unit is analogous to the Bodansky unit of phosphatase a~t i v i t y .~ In a group of 30 normal adults serum 5-nucleotidase activity has ranged from 0.3 to 3.2 units per 100 ml. of serum. Values do not appear to be influenced by age, sex, or race, and are fairly constant from week to week in the individual.Significant elevation of serum 5-nucleotidase activity has been observed thus far only in the presence of hepatobiliary disease. Normal 5-nucleotidase values + The work reported in this paper was supported in part by grants from the Detroit Receiving Hospital Research Corporation and the National Institute of Arthritis and Metabolic Diseases, Public Health Service, Bethesda, Md. 357(1) Substrate speci$city.(4) Eject of ethylenediaminefetraacetic acid (EDTA).(5) Electrophoretic mobility.

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