The catalytic subunit, gamma, of phosphorylase kinase is regulated by a complex set of interactions involving the calcium-binding protein calmodulin and two other subunits designated alpha and beta. These interactions regulate gamma activity that, at least for the calmodulin interactions, involves the regulatory domain in gamma spanning residues 302-366. Within this regulatory domain, we report the identification of a sequence (residues 326-334) that resembles the phosphorylation site in gamma substrates with the exception that a V residue (V332) occurs at the analogous position of the phosphorylated S/T residue. The inhibitory properties of the sequence were assayed with a 10-amino-acid peptide of the sequence. This peptide inhibits a truncated version of gamma, residues 1-300, which is missing the regulatory domain, more potently than it inhibits full-length gamma, and it is a better inhibitor of the full-length gamma at pH 8.2 than at pH 6.8. A similar peptide of the same sequence, except for a S substitution of the V residue, is a good substrate with a comparable Km and better Vmax than peptides of similar length that represent the phosphorylation site in the substrate of the enzyme, glycogen phosphorylase. A mutant gamma protein, with a S for V332 substitution ([V332S]gamma), was prepared using the baculovirus expression system. [V332S]gamma autophosphorylates by an intramolecular mechanism. This demonstrates that this sequence can occupy the catalytic site in the protein. Development of [V332S]gamma affords an experimental model in which the effects of the regulatory factors on autophosphorylation can be determined.