Altered expression and functional responses to cardiac β₃-adrenergic receptors (ARs) may contribute to progressive cardiac dysfunction in heart failure (CHF). We compared myocyte β₃-AR mRNA and protein levels and myocyte contractile, [Ca²⁺]_i transient, and Ca²⁺ current (I_Ca,L) responses to BRL-37344 (BRL, 10⁻⁸ mol/L), a selective β₃-AR agonist, in 9 instrumented dogs before and after pacing-induced CHF. Myocytes were isolated from left ventricular myocardium biopsy tissues. Using reverse transcription–polymerase chain reaction, we detected β₃-AR mRNA from myocyte total RNA in each animal. Using a cloned canine β₃-AR cDNA probe and myocyte poly A⁺ RNA, we detected a single band about 3.4 kb in normal and CHF myocytes. β₃-AR protein was detected by Western blot. β₃-AR mRNA and protein levels were significantly greater in CHF myocytes than in normal myocytes. Importantly, these changes were associated with enhanced β₃-AR–mediated negative modulation on myocyte contractile response and [Ca²⁺]_i regulation. Compared with normal myocytes, CHF myocytes had much greater decreases in the velocity of shortening and relengthening with BRL accompanied by larger reductions in the peak systolic [Ca²⁺]_i transient and I_Ca,L. These responses were not modified by pretreating myocytes with metoprolol (a β₁-AR antagonist) or nadolol (a β₁- and β₂-AR antagonist), but were nearly prevented by bupranolol or L-748,337 (β₃-AR antagonists). We conclude that in dogs with pacing-induced CHF, β₃-AR gene expression and protein levels are upregulated, and the functional response to β₃-AR stimulation is increased. This may contribute to progression of cardiac dysfunction in CHF.