The effect of glucagon administration on the heart glycogen synthetase and phosphorylase systems in control and insulin-pretreated rats has been studied. A rapid decrease in percent synthetase I activity was found in both control and insulin-pretreated animals after glucagon administration. This was associated with a simultaneous increase in percent phosphorylase a. Propranolol did not block the response. The results are compatible with a mechanism in which protein kinase is activated by increased cyclic AMP concentration which affects both the synthetase and phosphorylase systems simultaneously. The response of the synthetase and phosphorylase systems to glucagon was little affected by insulin; glycogen breakdown, however, was blocked completely. The mechanism of this is entirely unknown, but it appears to be due to an inhibition of phosphorylase a catalytic activity in vitro. Acetylcholine alone had no effect, nor did it modify the response of the synthetase and phosphorylase systems to either insulin or glucagon. However, it did produce a slight inhibition in percent phosphorylase a increase after glucagon.