Glycogen synthase kinase-5 (casein kinase-II) phosphorylates glycogen synthase on a serine termed site 5. This residue is just C-terminal to the 3 serines phosphorylated by glycogen synthase kinase-3, which are critical for the hormonal regulation of glycogen synthase in vivo. Although phosphorylation of site 5 does not affect the catalytic activity, it is demonstrated that this modification is a prerequisite for phosphorylation by glycogen synthase kinase-3. Since site 5 is almost fully phosphorylated in vivo under all conditions, the role of glycogen synthase kinase-5 would appear to be a novel one in forming the recognition site for another protein kinase.