In addition to the magnesium ion needed to form the true phosphate-donating substrate (ATP-Mg complex), we have determined that at least one additional Mg2+ ion is essential for the activation of protein tyrosine kinases. This activation was investigated in detail using purified Csk, Src, and the fibroblast growth factor receptor kinase, which led to the following conclusions. (1) The catalytic activity of these kinases is dependent on the Mg2+ concentration present in the assay, approaching saturation at 5-8 mM MgCl2, while ATP was saturated at approximately 1 mM MgCl2. (2) Extrapolation to zero free Mg2+ at a constant ATP-Mg concentration predicts zero activity, suggesting that free magnesium ion in excess of that needed to bind to ATP is essential for the activation of these enzymes. (3) The free magnesium ion activates Csk and Src kinase activity by increasing the Vmax but does not change their apparent Km(ATP-Mg). In contrast, the free magnesium ion activates the fibroblast growth factor receptor kinase activity by increasing its Vmax and decreasing its apparent Km(ATP-Mg). These and previous studies with the insulin receptor tyrosine kinase suggest that receptor-type protein tyrosine kinases respond to the concentration of free Mg2+ differently than soluble protein tyrosine kinases. (4) With the phosphate-accepting substrate as the variable ligand, increases in the concentration of free Mg2+ resulted in increases in the apparent Vmax for all tyrosine kinases examined, but the apparent Km response is dependent on the enzyme and the substrate used. While these studies do not pinpoint a single kinetic mechanism, they do suggest that additional magnesium ion(s) is(are) an essential activator for protein tyrosine kinases in addition to being a part of the ATP-Mg complex. The difference among protein tyrosine kinases in their kinetic response to the additional divalent metal cation and the potential biological significance of such are discussed.