During a study aimed at the specificity of binding of phosphorylase and glycogen synthase to glycogen particles we investigated the separation of these particles with the technique of high-performance liquid chromatography using several media. The technique allowed relatively rapid fractionations with little, if any, loss of enzyme activity. The basis of some separations was molecular weight of the particle. Of the molecular exclusion media used, Superose 6 had an exclusion limit large enough to accommodate most glycogens but excluded mouse liver and bovine liver glycogen particles. Toyopearl HW-75 F was a medium that could accommodate liver glycogen and gave us reasonably well-defined separation of the particles that carried phosphorylase and glycogen synthase. Separations on ion-exchange columns, that were obviously based on the overall charge of proteins associated with the glycogen particles, were not very effective in terms of allowing the separation of different types of particle. Of all the media used the hydrophobic interaction column was best in terms of separating what appeared to be distinct populations of particles that had definite preferences in terms of binding phosphorylase and glycogen synthase. As expected, these separations were based on the hydrophobic interaction of the proteins associated with the glycogen particles, since particles that had been deproteinized by a cold water extraction procedure did not bind to the column.