Glycogen synthase can be inactivated by sequential phosphorylation at the C-terminal residues Ser652 (site 4), Ser648 (site 3c), Ser644 (site 3b) and Ser640 (site 3a) catalysed by glycogen synthase kinase-3. In vitro, glycogen synthase kinase-3 action requires that glycogen synthase has first been phosphorylated at Ser656 (site 5) by casein kinase II. Recently we demonstrated that inactivation is linked only to phosphorylation at site 3a and site 3b, and that, in COS cells, modification of these sites can occur by alternative mechanisms independent of any C-terminal phosphorylations [Skurat and Roach (1995) J. Biol. Chem. 270, 12491-12497]. To address these mechanisms multiple Ser-->Ala mutations were introduced in glycogen synthase such that only site 3a or site 3b remained intact. Additional mutation of Arg637-->Gln eliminated phosphorylation of site 3a, indicating that Arg637 may be important for recognition of site 3a by its corresponding protein kinase(s). Similarly, additional mutation of Pro645-->Ala eliminated phosphorylation of site 3b, indicating a possible involvement of 'proline-directed' protein kinase(s). Mutation of Arg637 alone did not activate glycogen synthase as expected from the loss of phosphorylation at site 3a. Rather, mutation of both Arg637 and the Ser-->Ala substitution at site 3b was required for substantial activation. The results suggest that sites 3a and 3b can be phosphorylated independently of one another by distinct protein kinases. However, phosphorylation of site 3b can potentiate phosphorylation of site 3a, by an enzyme such as glycogen synthase kinase-3.