Rabbit skeletal muscle glycogen synthase contains multiple sites for phosphorylation. To investigate the relative importance of these sites, the enzyme was overexpressed in COS M9 cells, and Ser-->Ala mutations were introduced singly, or in combinations, at nine known phosphorylation sites. Overexpressed wild-type enzyme had a very low +/- glucose-6-P activity ratio of approximately 0.01, indicative that the glycogen synthase is in a highly phosphorylated state. No single Ser-->Ala mutation was able to cause a substantial increase in activity ratio; rather, simultaneous mutation at both NH2- and COOH-terminal sites was needed. The most effective combinations were mutations at site 3a (Ser-640) or site 3b (Ser-644) together with site 2 (Ser-7). The results were consistent with site 2 phosphorylation being a prerequisite for phosphorylation of site 2a (Ser-10). Mutation of site 5 (Ser-656) perturbed COOH-terminal phosphorylation but did not prevent inactivation. Expression of the most active mutants correlated with increased glycogen accumulation in the COS M9 cells. In summary, we conclude that (i) the sites most important for activating the enzyme are sites 2, 2a, 3a, and 3b; (ii) removal of phosphate from both NH2- and COOH-terminal sites is required for activation; and (iii) sites 3a and/or 3b can be phosphorylated in COS cells by mechanisms that do not depend on phosphorylation of site 5.