Although ligand-free, constitutive β₂-adrenergic receptor (AR) signaling has been demonstrated in naive cell lines and in transgenic mice overexpressing cardiac β₂-AR, it is unclear whether the dominant cardiac β-AR subtype, β₁-AR, shares the ability of spontaneous activation. In the present study, we expressed human β₁- or β₂-AR via recombinant adenoviral infection in ventricular myocytes isolated from β₁β₂-AR double knockout mice, creating pure β₁-AR and β₂-AR systems with variable receptor densities. A contractile response to a nonselective β-AR agonist, isoproterenol, was absent in double knockout mouse myocytes but was fully restored after adenoviral β₁-AR or adenoviral β₂-AR infection. Increasing the titer of adenoviral vectors (multiplicity of infection 10–1000) led to a dose-dependent expression of β₁- or β₂-AR with a maximal density of 1207 ± 173 (36-fold over the wild-type control value) and 821 ± 38 fmol/mg protein (69-fold), respectively. Using confocal immunohistochemistry, we directly visualized the cellular distribution of β₁-AR and β₂-AR and found that both subtypes were distributed on the cell surface membrane and transverse tubules, resulting in a striated pattern. In the absence of ligand, β₂-AR expression resulted in graded increases in baseline cAMP and contractility up to 428% and 233% of control, respectively, at the maximal β₂-AR density. These effects were specifically reversed by a β₂-AR inverse agonist, ICI 118,551 (10⁻⁷ M). In contrast, overexpression of β₁-AR, even at a greater density, failed to enhance either basal cAMP or contractility; the alleged β₁-AR inverse agonist, CGP 20712A (10⁻⁶ M), had no significant effect on basal contraction in these cells. Thus, we conclude that acute β₂-AR overexpression in cardiac myocytes elicits significant physiological responses due to spontaneous receptor activation; however, this property is β-AR subtype specific because β₁-AR does not exhibit agonist-independent spontaneous activation.