The Mg-dependent serine/threonine protein phosphatases, also known as type 2C phosphatases (PP2C), belong to a gene family distinct from the other serine/threonine phosphatases and tyrosine phosphatases. Here we report the purification to apparent homogeneity of a novel Mg-dependent, Ca-inhibitable serine/threonine protein phosphatase from bovine brain. It is a type 2C enzyme in view of its Mg requirement, resistance to okadaic acid and calyculin A, inability to use phosphorylase a as substrate, and a segment of amino acid sequence typical of all PP2C type phosphatases known to date. However, it differs from the other PP2C enzymes, particularly the mammalian PP2C and - isoforms, in that its molecular weight, 76,000, is considerably larger and that it is inhibited by Ca, NaF, and polycations, but not by orthovanadate. The Ca inhibition may not be related to its cellular regulation because of K values in the 20-90 µM range, but this property permits distinction of this enzyme from the other phosphatases. Although the precise physiological role of this phosphatase is not yet known, its ability to dephosphorylate a wide variety of phosphoproteins and its broad distribution, as shown by a survey of mouse tissues for its activity, suggest that it may serve an important cellular function.