Regulation of phosphorylase a formation in rabbit gracilis muscle in vivo was investigated by measurements of the responses of cyclic adenosine 3',5'-monophosphate (cyclic AMP), phosphorylase b kinase, and phosphorylase after administration of the catecholamine, isoproterenol. Responses to muscle contraction alone and in combination with isoproterenol were also measured. Phosphorylase a formation was stimulated without any changes in cyclic AMP or phosphorylase b kinase after administration of 4 x 10-12 moles of 1-isoproterenol. After 4 x 10-10 or 4 x 10-9 moles of 1-isoproterenol, cyclic AMP formation increased and nonactivated phosphorylase b kinase was converted to the activated form along with the stimulation of phosphorylase a formation. However, phosphorylase a formation preceded the stimulation of the formation of cyclic AMP and of activated phosphorylase b kinase. Three phases of phosphorylase a formation were identified: phosphorylase a formation without change in cyclic AMP and phosphorylase b kinase; phosphorylase a formation correlated with increased cyclic AMP and conversion of nonactivated phosphorylase b kinase to the activated form; further phosphorylase a formation with a greater cyclic AMP increase, but no greater formation of activated phosphorylase b kinase. Beta adrenergic blockade by 10-13 moles propranolol partially inhibited both cyclic AMP and phosphorylase a responses to 1-isoproterenol (4 x 10-10 moles) but not the formation of activated phosphorylase b kinase; i.e. phase 3 was inhibited. Tetanic electrical stimulation alone rapidly stimulated phosphorylase a formation without any change in cyclic AMP or phosphorylase b kinase. Tetanic electrical stimulation did not alter the responses to 4 x 10-10 moles of 1-isoproterenol. We conclude that the catalytic activity of nonactivated phosphorylase b kinase is sufficient for phosphorylase a formation during electrical stimulation and after isoproterenol administration. Therefore, phosphorylase a formation is not exclusively a consequence of an increase in cyclic AMP content and transformation of nonactivated phosphorylase b kinase to the activated form, but involves regulation by more complex mechanisms.