Evidence for active glycogen metabolism in normal mature red blood cells (RBC) is presented. Initial rates of 14C-U-glucose incorporation into erythrocyte glycogen were found to be independent of substrate concentration over a range of 3.3-16.6 mM. Incorporation of label into glycogen was initially linear but reached a plateau after a variable period of time that was inversely related to RBC concentration in the medium. The major part of the incorporated radioactivity resided in the outer branches of the glycogen molecule. The optimum pH for 14C-U-glucose incorporation into glycogen was pH 7.6. Replacing the radioactive glucose employed for incorporation after 1 hr of incubation with nonlabeled glucose resulted in a gradual loss of radioactivity from erythrocyte glycogen. In normal cells, glycogen synthesis and breakdown do not result in any significant accumulation of glycogen, whereas in erythrocytes with enzyme defects affecting glycogen breakdown, substantial deposition of glycogen may be observed.