Phosphorylase removes glucosyl units from the terminal branches of glycogen through phosphorolysis, forming glucose-1-P. It is present in two interconvertible forms, phosphorylaseaandb. Theaform is the active form and is rate limiting in glycogen degradation. The activities of phosphorylaseaand of total phosphorylase as conventionally measured exceed the activities of glycogen synthase R (active form) and of total synthase by ∼10- and 20-fold. Thus, unless phosphorylaseais inhibited or compartmentalized or its substrates are exceedingly lowin vivo,net glycogen synthesis could not occur. In addition, following an administered dose of glucose, phosphorylaseaactivity changes little when glycogen is being synthesized, is stable, or is being degraded, suggesting an important role for allosteric effectors in regulation. Therefore, we have determined the effect of potential modifiers of enzyme activity at estimated intracellular concentrations. Purified liver phosphorylaseawas used. Activity was measured in the direction of glycogenolysis, at 37°C, pH 7.0, and under initial rate conditions. Both aK_mand a near-saturating concentration of inorganic phosphate (substrate) were used in the assays. A physiological concentration of AMP was saturating. It decreased theK_mfor P_iby ∼50% and stimulated activity. ADP, ATP, and glucose inhibited activity. Fructose-1-P inhibited activity only at a high and nonphysiological concentration. Glucose-6-P and UDP–glucose were not significant inhibitors. Inhibition of activity by ADP was little affected by the addition of AMP. However, AMP partially abolished the inhibitory effect of ATP and completely abolished the inhibitory effect of glucose. When AMP, ADP, ATP, glucose-6-P, UDP–glucose, glucose, and fructose-1-P were added together, the net effect was no change in phosphorylaseaactivity compared to the activity without any effectors. In addition, changes in glucose concentration did not affect activity. K glutamine modestly stimulated activity. Numerous other metabolites were tested and were without effect. The present data indicate that the known endogenous allosteric effectors cannot explain the smaller than expectedin vivophosphorylaseaactivity or the regulation of phosphorylaseaactivity.