Purified sarcolemmal and light vesicle (intracellular) fractions of β-adrenergic receptors were used to examine the effects of propranolol on receptor translocation in guinea pig heart. Guinea pigs were given propranolol (0.15 mg/kg/hr) via minipumps for 7 days and either killed or made ischemic for 1 hour via a coronary ligature. Propranolol treatment led to an externalization of β-receptors from light vesicle to sarcolemmal fractions. This externalization increased the number of surface β-adrenergic receptors that were functional, as assessed by isoproterenol-stimulated adenylate cyclase activity. After chronic propranolol treatment, ischemia did not further alter receptor distribution. These results suggest that externalization of β-adrenergic receptors from a light vesicle fraction to the sarcolemma contributes to up-regulation of β-receptors that occur in response to both propranolol treatment and ischemia. Because propranolol-treated animals show blunting in externalization after myocardial ischemia, propranolol treatment and myocardial ischemia appear to access the same pool of intracellular β-adrenergic receptors. Depletion of this pool of receptors along with receptor blockade may thus contribute to the mechanism by which the drug is efficacious in preventing some adverse effects of ischemia.