Heart failure (HF) is a significant cause of mortality in the aged. Perturbations to calcium (Ca2+) homeostasis in failing ventricular myocytes have been well documented. However, the transmural variation in Ca2+ homeostasis of the failing left ventricle is incompletely understood and data on how this is influenced by ageing is lacking. The aim of this work was to identify any changes in transmural excitation-contraction coupling that may elucidate potential arrhythmogenic mechanisms in the aged failing heart. HF was induced by tachypacing aged (over 8 years of age) Welsh Mountain sheep anaesthetised using isoflurane (1-3%) and analgesia was provided with meloxicam (0.5mg/kg s/c). Single isolated left ventricular myocytes from either the epicardium or endocardium were voltage clamped using the perforated patch technique at 37 °C. Cells were loaded with the Ca2+ sensitive indicator Fluo-5F AM in order to measure changes to intracellular Ca2+ concentration. Data are presented as mean ± standard error of the mean (SEM). Differences between groups were determined using a two-way ANOVA. In HF, Ca2+ transient amplitude was decreased in the epicardium (41 ± 17%; n = 12 – 18, p = 0.023), but not in the endocardium when compared to control levels. A reduction in epicardial peak L-type calcium current (ICa-L) density at +10 mV (37 ± 8%; n = 13 – 17, p < 0.001) was found in the epicardial failing myocytes, along with a decrease in total sarcoplasmic reticulum (SR) Ca2+ content (39 ± 9%; n = 11, p = 0.014). However, no changes to either the ICa-L density or SR Ca2+ content were found in endocardial cells. Threshold SR Ca2+ content was determined by using 10 mM Ca2+ in external solutions to induce Ca2+ waves. Under these conditions, in failing epicardial myocytes only, SR Ca2+ content increased by 217 % from 31 ± 3 μmol/l to a threshold level of 100 ± 10 μmol/l (n = 6 – 11, p < 0.001). However, when 100 nM isoprenaline was applied to the failing epicardial cells SR Ca2+ content was found to be 83 ± 7 μmol/l and Ca2+ waves developed at 121 ± 8 μmol/l which was not different from baseline (n = 4 – 5, p = 0.144). The smaller Ca2+ transient amplitude in failing epicardial cells was the result of a smaller peak ICa-L density and reduced SR Ca2+ content. Under control conditions, in failing epicardial cells, SR Ca2+ content is lower than the threshold at which Ca2+ waves develop. However, with the application of isoprenaline SR Ca2+ content is similar to threshold levels making these cells under β-adrenergic stimulation more likely to develop spontaneous Ca2+ release, potentially resulting in arrhythmias.