The regulation of glycogen phosphorylase and glycogen breakdown in human skeletal muscle has been investigated using the needle biopsy technique. Preliminary studies showed that the activity of phosphorylase in vitro was dependent upon the concentration of inorganic phosphate (Pi) used in the assay system. The Km of phosphorylase a for Pi was found to be 26.2 mmol/l, and that of (a+b) (assayed in the presence of saturating AMP) was 6.8 mmol/l. Because of the difference in Km the apparent percentage of a to (a+b) activity varies with the Pi concentration used in the assay system. Phosphorylase a and (a+b) activities were therefore adjusted to saturating Pi concentrations. The ratio of the activities in this case is independent of the Pi concentration and constitutes a minimal estimate of the fraction of phosphorylase molecules in the a form. The fraction of phosphorylase in the a form in resting muscle was as a mean 22%. Despite nearly a quarter of the phosphorylase being in the a form glycogenolytic activity is extremely low. It is proposed that the concentration of Pi at the active site of the enzyme is low compared to the Km for this of either form of the enzyme, and is limiting to activity. A Pi concentration in resting muscle of 1-3 mmol/l was calculated. During epinephrine infusion at rest 90% of the phosphorylase was transformed to the a form but only a moderate increase in the glycogenolytic rate occurred. This rate approximated to 5-10% of the maximum rate of the enzyme (Vmaxa). During prolonged epinephrine infusion the glycogenolytic rate decreased despite the continuance of 90% or more of the phosphorylase in the a form. In contrast to epinephrine infusion prolonged ischemia resulted in a decrease in the mole fraction of phosphorylase a and simultaneously in an increase of the glycogenolytic rate. During isometric and dynamic exercise there was a rapid transformation of phosphorylase b to a paralleled by pronounced increase in the rate of glycogen breakdown. The increased rate of glycogenolysis during isometric exercise was close to the Vmax of phosphorylase a in vivo. When either form of exercise was continued to fatigue/exhaustion, a re-transformation of phosphorylase a to b was observed. During dynamic exercise cAMP in the muscle increased two fold. This increase was blocked by the prior administration of propranolol.+