Cardiomyocytes express both β₁- and β₂-adrenergic receptors, and these receptors play a differential role in chronotropic and inotropic effects of the heart. Caveolae are known as an important regulator of G-protein-coupled receptor signaling. In the present report, we examined whether caveolae have a role in β-adrenergic receptor-stimulated cAMP production and protein kinase A activation in neonatal myocytes. Isoproterenol-stimulated cAMP production was mediated by β₁- and β₂-subtypes, which depends on the receptor number of each subtype. However, protein kinase A activation was exclusively mediated by the β₁-subtype. Disruption of caveolae by methyl-β-cyclodextrin treatment did not affect the relative contribution of subtypes to isoproterenol-stimulated cAMP production. β₁-Subtype-mediated protein kinase A activation was also not affected by the disruption of caveolae. These results suggest that β₁-adrenergic receptor-mediated protein kinase A activation is compartmentalized and independent of caveolae.