Phosphorylase kinase (PhK) and truncated γ subunit, denoted γ1–300, can phosphorylate seryl andtyrosyl residues dependent on the metal ion [Yuan, C.-J., Huang, C. F., & Graves, D. J. (1993) J. Biol. Chem. 268, 17683–17686]. Recombinant γ1–300 was used to explore its dual specificity and the location of the metal ion binding sites by using site-directed mutagenesis. Two approaches were taken to generate 26 mutants. First, on the basis of the crystal structure of cAMP-dependent protein kinase (cAPK), the invariant Asn155 and highly conserved Asp168-Phe169-Gly170 residues were mutated. Changes included production of N155H, D168E, D168N, F169R, G170V, G1701, G170L (less than 1% of enzymatic activities were found in these mutants), F169W, and G170A mutants. Second, charge to alanine and charge reversal scanning mutations were used to probe the metal ion binding sites. Two mutants, E111K and E154R, showed very different metal ion response compared to wild-type γ and were further characterized. The mutants F169W, G170A, E111K, and E154R had 15%, 5%, 8%, and 25% specific activity relative to wild-type γ, respectively. The folding pattern of wild-type and mutated enzyme forms of γ was determined by photoacoustic infrared spectroscopy. Conformational disruptions were found in G170V, G170I, and G170L mutants, but the conformation of the rest of the mutants was similar to that of wild-type γ, suggesting that the loss of enzymatic activities of these mutants was not because of incorrect refolding. Kinetic analyses of mutants indicate that Asn155 and Asp168 residues influence maximal velocity and that Glu111, Glu154, Phe169, and Gly170 residues influence binding of MgATP and phosphorylase b. The interactions of divalent cations, Mg2+ and Mn2+, with E111K, E154R, N155H, F169W, and G170A mutants were different from those with the wild-type, suggesting that Glu111, Glu154, Asn155, Phe169, and Gly170 contribute to the character of the metal ion binding sites. Our results suggest that the metal ion binding sites reside between the D168FG loop and the E111-KPE154N loop, similar to the metal ion binding sites in cAPK. The tyrosine kinase activity of γ1–300 in the presence of Mn2+ was increased in E154R (251%), unchanged in E111K, F169W, and G170A, and reduced in N155H (5%). Typically, the mutations had a more pronounced effect on serine kinase activity than on tyrosine kinase activity. The activity ratio (tyrosine kinase activity to serine kinase activity) increased in the mutants, suggesting that these five residues have different roles in the two activities. Our results support the view that different conformational states induced by metal ions are important for dual specificity.