The regulatory-targeting subunit (R_GL, also called G_M) of the muscle-specific glycogen-associated protein phosphatase PP1G targets the enzyme to glycogen where it modulates the activity of glycogen-metabolizing enzymes. PP1G/R_GL has been postulated to play a central role in epinephrine and insulin control of glycogen metabolism via phosphorylation of R_GL. To investigate the function of the phosphatase, R_GLknockout mice were generated. Animals lacking R_GL show no obvious defects. The R_GL protein is absent from the skeletal and cardiac muscle of null mutants and present at ∼50% of the wild-type level in heterozygotes. Both the level and activity of C1 protein are also decreased by ∼50% in the R_GL-deficient mice. In skeletal muscle, the glycogen synthase (GS) activity ratio in the absence and presence of glucose-6-phosphate is reduced from 0.3 in the wild type to 0.1 in the null mutant R_GL mice, whereas the phosphorylase activity ratio in the absence and presence of AMP is increased from 0.4 to 0.7. Glycogen accumulation is decreased by ∼90%. Despite impaired glycogen accumulation in muscle, the animals remain normoglycemic. Glucose tolerance and insulin responsiveness are identical in wild-type and knockout mice, as are basal and insulin-stimulated glucose uptakes in skeletal muscle. Most importantly, insulin activated GS in both wild-type and R_GLnull mutant mice and stimulated a GS-specific protein phosphatase in both groups. These results demonstrate that R_GL is genetically linked to glycogen metabolism, since its loss decreases PP1 and basal GS activities and glycogen accumulation. However, PP1G/R_GL is not required for insulin activation of GS in skeletal muscle, and rather another GS-specific phosphatase appears to be involved.