The AMP-activated protein kinase (AMPK) is heterotrimer consisting of α catalytic subunit and β/γ regulatory subunits. It acts as a critical focal point for whole body and cellular mechanisms maintaining energy homeostasis by regulating carbohydrate and lipid metabolism, food intake, gene transcription, and protein synthesis. The AMPK β subunit contains a glycogen-binding domain that has been shown to associate with glycogen particles in vitro and glycogen phosphorylase and glycogen synthase in cultured cells. To determine whether AMPK associates with glycogen particles in vivo, we developed a procedure to purify glycogen α-particles to apparent homogeneity from rat liver. Using immunoreactivity and mass spectrometry we determined that AMPK does not associate with the glycogen particle in livers from random-fed rats. This surprising finding indicates that the glycogen-binding properties of the AMPK β subunit are likely regulated and responsive to the metabolic status of the hepatocyte.