For over four decades free Mg 21 ions, that is, those in excess of MgATP, have been reported to affect a wide variety of properties of phosphorylase kinase (PhK), including its affinity for other molecules, proteolysis, chemical crosslinking, phosphorylation, binding to certain monoclonal antibodies, and activity, which is stimulated. Additionally, for over three decades Mg 21 has been known to act synergistically with Ca 21 , another divalent activator of PhK, to affect even more properties of the enzyme. During all of this time, however, no study has been performed to determine the overall effects of free Mg 21 ions on the physical properties of PhK, even though the effects of Ca 21 ions on PhK's properties are well documented. In this study, changes in the physicochemical properties of PhK induced by Mg 21 under nonactivating (pH 6.8) and activating (pH 8.2) conditions were investigated by circular dichroism spectroscopy, zeta potential analyses, dynamic light scattering, second derivative UV absorption, negative stain electron microscopy, and differential chemical crosslinking. The effects of the activator Mg 21 on some of the properties of PhK measured by these techniques were found to be quite different at the two pH values, and displayed both differences and similarities with the effects previously reported to be induced by the activator Ca 21 (Liu et al., Protein Sci 2008;17:2111-2119. The similarities may reflect the fact that both cations are activators, and foremost among their similarities is the dramatically less negative zeta potential induced by their binding to PhK.