The glycogen phosphorylase of Dictyostelium discoideum has been purified over 200-fold from cells in the culmination stage of development. Analytical gel electrophoresis of the purified enzyme indicates one major protein band with a molecular weight of approximately 210,000. Gel elution verified the presence of phosphorylase activity associated with the protein band. Electrophoresis of partially purified extracts prepared from amoebae cells revealed the absence of phosphorylase protein. Sodium dodecyl sulfate electrophoresis on 6% gels indicated that the purified phosphorylase is composed of subunits, 95,000 in molecular weight. The purified enzyme exhibited normal Michaelis-Menten kinetics and activity was not stimulated by added nucleotides such as 5'-AMP. Nucleotide sugars (GDP-glucose, UDP-glucose, ADP-glucose) were competitive inhibitors of the phosphorylase reaction.