Nucleoside diphosphatase from rat liver microsomes was purified about 3,000-fold to a homogenous state using a procedure including Concanavalin A-Sepharose and phenyl-Sepharose column chromatographies. The molecular weight was estimated as 130,000 +/- 2,000 for the native enzyme. Sodium dodecyl sulfate gel electrophoresis indicated the presence of two identical subunits. The purified enzyme was confirmed to be a glycoprotein containing approximately 9% carbohydrate consisting of mannose (4.2%). On the amino acid analysis, the enzyme contained 27.1% acidic, 10.7% basic and 34.6% non-polar amino acids. The isoelectric point of 4.85 with the enzyme would, at least partly, be ascribed to the higher content of glutamic acid. While the activity of the purified enzyme was inhibited by EDTA and o-phenanthroline, the inhibited enzyme activity was restored by the addition of Mg2+, Ca2+, Mn2+, Co2+ or Zn2+. The enzyme was found to be a metalloenzyme containing approximately 2 atoms or less of Zn2+ per molecule of the native enzyme.