This study identifies a 100-residue domain within the rabbit skeletal muscle regulatory subunit (PP1G) that binds both type-1 protein phosphatase (PP1C) and glycogen. An N-terminal portion of PP1G was cloned by RT-PCR, and different sized fragments were expressed in bacteria as glutathione S-transferase (GST) fusion proteins. A GST−PP1G fusion containing residues 51−240 bound both PP1C and glycogen, whereas GST alone or fusions containing residues 51−140 or 241−360 bound neither PP1C nor glycogen. The PP1C in whole cell lysates or partially purified PP1C from skeletal muscle, or a complex of PP1C−MCLR−biotin, all bound more effectively than Mn²⁺-activated, recombinant PP1C purified from bacteria. Binding was enhanced by increasing the ionic strength and was disrupted by ethylene glycol, consistent with hydrophobic interactions being critical for stable association. Phosphorylation of the GST−PP1G fusion by cAMP-dependent protein kinase prevented completely association of PP1C. This domain of PP1G, from residues 141−240, contains two sequence motifs of hydrophobic residues:  Gx₈FEKx₁₀W and DxFxFxIxL, that are conserved among the known glycogen-binding PP1 regulatory subunits. These segments are predicted to form an α helix and a β sheet, and we propose that they are the sites for association with PP1C and glycogen, respectively.