1. Pyruvate-dehydrogenase phosphatase was extensively purified from pig heart muscle. 2. The molecular weight was determined to be in the range between 92000 and 95000. By dodecylsulfate-gel electrophoresis there was no indication for a subunit structure of the protein.3. The phosphatase is dependent on Mg2f or Mn2+. Half-maximal activity was obtained, in histidine buffer, a t 2.5 mM MgC1, and 1.8 mM MnCI,, respectively.4. Fluoride a t a concentration of 0.6mM inhibited phosphatase activity by 50°/, in the presence of Mg2+, but not of Mn2+. The enzyme was not affected by alkylating thiol-reagents.5. Studies with sequestering agents suggest that pyruvate-dehydrogenase phosphatasc contains a metal which, besides Mg2+ or Mn2+, is essential for its function.The purified enzyme complex of mammalian pyruvate dehydrogenase is converted from the catalytically active form to the inactive form by ATPdependent phosphorylation [l --51. The enzyme responsible for the phosphate incorporation was called "pyruvate dehydrogenase kinase". Reactivation of the phospho pyruvate-dehydrogenase complex is catalyzed by a second enzyme entity, which was designated pyruvate-dehydrogenase phosphatase, since the pyruvate-dehydrogenase complex is concomitantly dephosphorylated during reactivation [ 1 , 3 -51.