Phosphorylase kinase is a complex enzyme in both structure and regulation. It has a subunit stoichiometry of ((alpha)(beta)(gamma)(delta))(,4), and the (alpha), (beta), and (delta) subunits have known regulatory functions, whereas the (gamma) subunit has a catalytic site. The (gamma) subunit, separated from the holoenzyme by reversed-phase high-performance liquid chromatography (HPLC), had no activity in the usual assay mixture, but was reactivated in the presence of Ca('+2) and calmodulin (which is identical to the (delta) subunit). The renaturation and reactivation process, which is totally dependent on Ca('+2) and calmodulin, is optimized under specific conditions of temperature, time, pH, the presence of a stabilizing protein such as bovine serum albumin or, and the concentrations of HPLC solvents and the (gamma) subunit. The reactivated (gamma) subunit, still in the presence of calmodulin, shared several properties with a (gamma)(delta) complex previously isolated, including the strong, Ca('+2)-independent nature of the (gamma)-calmodulin bond. The (gamma) subunit free of calmodulin was obtained by reactivation of the HPLC-isolated (gamma) subunit with calmodulin bound covalently to agarose. After reactivation, the (gamma) subunit was eluted from the calmodulin-agarose with a solution containing 1.0 M Tris (pH 7.0), 1% Triton X-100, 1 mM EGTA, and 5 mM dithiothreitol. The isolated (gamma) subunit is catalytically active, but, unlike the holoenzyme activity, the (gamma) subunit is totally independent of Ca('+2), although it can be activated by Ca('+2) plus calmodulin. Apparent Km values of the (gamma) subunit for the substrates ATP and phosphorylase b are equivalent to those found for the activated form of phosphorylase kinase, but Vm values are only about 10% of those of other forms of the enzyme. Unlike the (gamma)(delta) complex, the (gamma) subunit is pH dependent in the range of 6.8 to 9.0. Free Mg('+2) stimulates (gamma) subunit activity, whereas free Mn('+2) is inhibitory. ADP is a competitive inhibitor with a Ki of 60 (mu)M. Finally, the (gamma) subunit was shown to phosphorylate many phosphorylase kinase substrates, including troponin I, troponin T, (kappa)-casein, and myelin basic protein.