The regulatory mechanism of a phosphoprotein phosphatase [EC 3.1.3.16], which is considered to catalyze the dephosphorylation reaction of several phosphoproteins (glycogen synthetase-D [EC 2.4.1.11], phospho-form of phosphorylase b kinase [EC 2.7.1.38], phosphohistone and phosphorylase a [EC 2.4.1.1]), was studied with partially purified preparations from rabbit skeletal muscle. Time- and temperature-dependent inactivation and reactivation of phosphohistone phosphatase, as well as phosphorylase phosphatase [EC 3.1. 3.17], were observed on pre-incubation of the enzyme(s) with ATP, and subsequent incubation with divalent metal ions (Mg2+, Mn2+, or Co2+) without any change of molecular size. Manganese, however, instantly restored the activity of the ATP-inactivated enzyme, and increased the maximal velocity of the enzyme while decreasing its affinity to phosporylase a. However, the metal ion inhibited the reactivated enzyme competitively with respect to phosphorylase a. It is suggested that phosphoprotein phosphatase(s) is a metalloenzyme, and that ATP results in a conformational change of the enzyme protein in such a way that a metalion can be easily released due to the chelating effect of ATP, or incorporated (in the presence of exess metal ions) into the enzyme protein.