The glucosylation site on glycogenin, the protein primer required for de novo glycogen synthesis, has been identified. The glucose is attached at position C1 in a glycosidic linkage with a unique tyrosine, and the sequence surrounding this residue was found to be: His-Leu-Pro-Phe-Ile-Tyr-Asn-Leu-Ser-Ser-Ile-Ser-Ile-Tyr(Glc)-Ser-Tyr-Leu -Pro- Ala-Phe-Lys. The same tyrosine residue is glycosylated whether glycogenin is isolated as a complex with the catalytic subunit of glycogen synthase, or covalently attached to glycogen. The possibility that insulin and growth factors may enhance glycogen synthesis via stimulation of the priming reaction is discussed.