Protein phosphatase-2C (PP-2C), an Mg2+-dependent enzyme, is one of four protein phosphatase catalytic subunits in the cytoplasm of mammalian cells that are capable of dephosphorylating a variety of regulatory proteins. The enzyme can be purified from skeletal muscle and other mammalian tissues. This chapter describes the procedures for the purification. PP-2C is most conveniently assayed by its ability to dephosphorylate casein, although other substrates, such as phosphorylase kinase and myosin P-light chain, can also be used. PP-2C cannot be assayed reliably at early stages of the preparation, because it represents only a very small proportion of the phosphatase activity toward casein and most other substrates. The major casein phosphatase activity in tissue extracts is protein phosphatase-2A (PP-2A). The purification procedure should be applicable to the isolation of PP-2C from all mammalian tissues.