Reduced inotropic responsiveness is characteristic of heart failure (HF). This study determined the cellular Ca 2+ homeostatic and molecular mechanisms causing the blunted β-adrenergic (β-AR) response in HF. We induced HF by tachypacing in sheep; intracellular Ca 2+ concentration was measured in voltage-clamped ventricular myocytes. In HF, Ca 2+ transient amplitude and peak L-type Ca 2+ current (I Ca-L) were reduced (to 70 ± 11% and 50 ± 3.7% of control, respectively, P <0.05) whereas sarcoplasmic reticulum (SR) Ca 2+ content was unchanged. β-AR stimulation with isoprenaline (ISO) increased Ca 2+ transient amplitude, I Ca-L and SR Ca 2+ content in both cell types; however, the response of HF cells was markedly diminished (P <0.05). Western blotting revealed an increase in protein phosphatase levels (PP1, 158 ± 17% and PP2A, 188 ± 34% of control, P <0.05) and reduced phosphorylation of phospholamban in HF (Ser16, 30 ± 10% and Thr17, 41 ± 15% of control, P <0.05). The β-AR receptor kinase GRK-2 was also increased in HF (173 ± 38% of control, P <0.05). In HF, activation of adenylyl cyclase with forskolin rescued the Ca 2+ transient, SR Ca 2+ content and SR Ca 2+ uptake rate to the same levels as control cells in ISO. In conclusion, the reduced responsiveness of the myocardium to β-AR agonists in HF probably arises as a consequence of impaired phosphorylation of key intracellular proteins responsible for regulating the SR Ca 2+ content and therefore failure of the systolic Ca 2+ transient to increase appropriately during β-AR stimulation. © 2011 The Authors. Journal compilation © 2011 The Physiological Society.