Phosphorylase kinase purified from rabbit skeletal muscle has a mol. wt. of 1.3 x 10('6) and comprises four different subunits, ((alpha)(beta)(gamma)(delta))(,4). By dissociating the holoenzyme with 1.8 M LiBr and subsequent gel filtration, ion-exchange chromatography or blue-Dextran Sepharose 4B affinity chromatography, two catalytically active, monomeric, and stoichiometric species, (alpha)(gamma)(delta) and (gamma)(delta) complexes, were isolated to apparent homogeneity. The molecular weights, Stokes radii, and sedimentation coefficients are estimated to be 243000, 53.2 (ANGSTROM), and 11.8S, respectively, for the (alpha)(gamma)(delta) complex, and 66000, 35.2 (ANGSTROM), and 4.2S, respectively, for the (gamma)(delta) complex. Individual (alpha), (beta), and (gamma) subunits were purified and their amino acid compositions analyzed. Other physicochemical parameters such as the UV absorption spectrum and circular dichroism were also investigated for the (alpha)(gamma)(delta) complex and compared to the holoenzyme;The catalytic properties of the holoenzyme, (alpha)(gamma)(delta) and (gamma)(delta) complexes are distinct from one another in their pH 6.8/8.2 activity ratios, linearity in reaction progress curves, and sensitivity to Ca('2+). The molar activities estimated for the holoenzyme, the (alpha)(gamma)(delta) and (gamma)(delta) complexes are ca. 99, 91, and 104 molecules s('-1), indicating that the (gamma) subunit may contain the sole catalytic site for the conversion of phosphorylase b. The three forms of kinase are regulated differentially by glycogen, heparin, exogenous calmodulin, and 1,2-dimethoxyethane, suggesting that the (alpha) or (beta), or both, subunits may respond differently to these effectors. Limited proteolysis of the (alpha) subunit activated the (alpha)(gamma)(delta) complex, whereas phosphorylation of the (alpha) subunit has no effect on the kinase activity;Affinity chromatography on calmodulin-Sepharose 4B column suggests that the (alpha) subunit is responsible for the binding of the exogenous calmodulin. The intrinsic (delta) subunit interacts tightly with the (alpha) and (gamma) subunits and there seemed to be a trigonal relationship for the interaction of these three subunits. A model for a possible structure of phosphorylase kinase is proposed;The substrate specificity of phosphorylase kinase was also investigated by using synthetic substrates and compared to cAMP-dependent protein kinase, insofar as possible.