The γ-subunit of phosphorylase kinase contains a protein kinase catalytic domain (residues 20-276) and a regulatory domain (residues 276-386). The purpose of the present investigation was to develop monospecific antibodies against four synthetic γ-subunit regulatory domain peptides (PhK1: 362-386; PhK5: 342-366; PhK9: 322-346; PhK13: 302-326) to use as probes to study the structure of the regulatory domain. Each affinity-purified antibody was characterized with regard to its ability to bind three different structural forms of the γ-subunit: the isolated γ-subunit, the γ-δ complex, and the holoenzyme complex (αβδγ) 4 . Of the four antibodies, binding of affinity-purified anti-PhK13 was most affected by alterations in γ-subunit interactions. Taken together, the data from this investigation indicate that the regulatory domain of the γ-subunit can assume different immunochemically distinguishable conformations as the result of interactions among the α-, β-, γ-, and δ-subunits of phosphorylase kinase.