Targeted cardiac overexpression of the α-subunit of the heterotrimeric G protein G_q in transgenic mice evokes hypertrophy and depressed stimulation of cardiac inotropy and chronotropy by β-adrenergic receptor (βAR) agonists in vivo, which is a hallmark of many forms of experimental and human heart failure. The molecular basis of this βAR dysfunction was explored in transgenic mice overexpressing G_αq ∼5-fold over background. Isoproterenol-stimulated adenylyl cyclase activities in myocardial membranes were significantly depressed in G_αqmice compared with nontransgenic controls (19.7 ± 2.6 versus 43.7 ± 5.6 pmol/min/mg) without a decrease in βAR expression levels. Functional coupling of both βAR subtypes was impaired. Similarly, in whole-cell patch-clamp studies, βAR stimulation of L-type Ca²⁺ channel currents was depressed ∼75% in the G_αq mice. Cardiac βAR from these mice showed decreased formation of the active high-affinity conformation (R _H = 29% versus 62% for nontransgenic littermates), confirming a receptor-G_s-coupling defect. Of the three candidate kinases that might impose this uncoupling by receptor phosphorylation (protein kinase A, βAR kinase, protein kinase C), only protein kinase C activity was elevated in G_αq mouse hearts. Type V adenylyl cyclase was decreased ∼45% in these mice, consistent with decreased basal, NaF, and forskolin-stimulated enzyme activities. Although cellular G_s levels were unaltered, G_i2 and G_i3 were increased in G_αq mice. Pertussis toxin treatment of isolated G_αq myocytes resulted in an improvement in βAR, but not that of forskolin or NaF, stimulation of adenylyl cyclase. Thus three distinct mechanisms contribute to impaired βAR function by in vivo G_q signaling cross-talk in myocytes. Because many elements of hypertrophy and/or failure in cellular and animal models can be initiated by increased G_αq signaling, the current work may be broadly applicable to interfaces whereby modification of heart failure might be considered.