Catecholaminergic activation of myocardial -adrenergic receptors (AR) is the principle mechanism regulating cardiac function. Agonists desensitize AR through G protein-coupled receptor kinase-mediated uncoupling and -arrestin-mediated internalization. Although inhibition of myocardial G protein-coupled receptor kinase-2 enhances cardiac function and reverses heart failure, pathophysiological effects of modulated AR internalization͞re-cycling are unknown. We used mutation and transgenic expression of Rab4, which regulates vesicular transport of heptahelical receptors to plasma membranes, to interrogate in vivo AR trafficking and cardiac function. Expression of constitutively active Rab4 Q72L had no effects on cardiac structure or function, but dominant inhibitor Rab4 S27N impaired responsiveness to endogenous and exogenous catecholamines. To relate AR trafficking to diminished cardiac function, Rab4 mutant mice were crossbred with mice overexpressing human 2AR. In unstimulated 2AR overexpressors, 2AR localized to heavier endosomes and translocated to lighter, caveolin-rich fractions after isoproterenol stimulation. Coexpression of 2AR with activated Rab4 Q72L caused loss of receptors from heavier endosomes while retaining normal inotropy. In contrast, coexpression of 2AR with inhibitory Rab4 S27N mimicked isoproterenol-induced receptor redistribution to caveolae, with diminished cardiac inotropy. Rab4 inhibition alone prevented resensitization after isoproterenol-induced in vivo adrenergic desensitization. Confocal and ultrastructural analyses revealed bizarre vesicular structures and abnormal accumulation of 2AR in the sarcoplasm and subsarcollema of Rab4 S27N, but not Q72L, mice. These data provide evidence for constant bidirectional sarcollemal-vesicular AR trafficking in the in vivo heart and show that Rab4-mediated recycling of internalized AR is necessary for normal cardiac catecholamine responsiveness and resensitization after agonist exposure.