Alterations in cardiac excitation-contraction (EC) coupling are known to occur during heart failure (HF). We therefore sought to characterise EC coupling and investigate the responses to β-adrenergic stimulation in an ovine model of HF. Sheep aged 18 months were subjected to right ventricular tachypacing (210bpm) to induce heart failure with untreated age-matched animals used as controls. Animals were anaesthetised with isoflurane (1-3% in oxygen) and perioperative analgesia provided (meloxicam 0.5 mg/kg). Myocytes were isolated from the left ventricular mid-myocardial wall and whole cell voltage clamp experiments performed at 37°C using a Cs+ based pipette solution containing the Ca indicator Fluo-5F. Western blots were performed using left-ventricular wall protein extracts from five control and seven HF animals. Data are presented as mean ± S.E.M with the Student’s t-test or Mann-Whitney U-test used for statistical analyses. A reduction in the amplitude of ICa-L occurred in HF (3.97± 0.23 pA/pF v 8.02±0.42 pA/pF, n= 42-47 cells). This was accompanied by a decrease in the amplitude of the systolic Ca transient (87±12.6 nM v 130±12.6 nM, n=22 cells). SERCA function was reduced by 31% whereas NCX function increased by 42%. SR Ca content was unchanged in HF. A reduction in phospholamban phosphorylation was observed at both Ser16 (70%) and Thr17 (52%) sites. β-adrenergic stimulation (100 nM isoprenaline) normalised ICa-L in HF animals, comparable to control conditions in healthy sheep (8.35±1.02 pA/pF, n=27 cells). Ca transient amplitude, SR Ca content and SERCA function were all increased by isoprenaline in HF but to a substantially smaller extent than occurred in controls. Bypassing β-adrenergic receptor activation with forskolin (3 µM) increased Ca transient amplitude to an extent similar to isoprenaline-stimulated activity in healthy sheep (576±152 nM v 623±104 nM, n=6-17 cells). Western blotting additionally revealed a 70% increase in the expression of the β-adrenergic receptor kinase (GRK2) in HF. Ventricular tachypacing in sheep produces multiple abnormalities in cardiac EC coupling, notably a reduced responsiveness to β-adrenergic stimulation. Our data suggests this is a result of a loss of β-adrenergic receptors rather than perturbed PKA- dependent effects.