Heart failure is associated with pump dysfunction and remodeling but it is not yet known if the condition affects different transmural regions of the heart in the same way. We tested the hypotheses that the left ventricles of non-failing human hearts exhibit transmural heterogeneity of cellular level contractile properties, and that heart failure produces transmural region-specific changes in contractile function.Permeabilized samples were prepared from the sub-epicardial, mid-myocardial, and sub-endocardial regions of the left ventricular free wall of non-failing (n=6) and failing (n=10) human hearts. Power, an in vitro index of systolic function, was higher in non-failing mid-myocardial samples (0.59±0.06μWmg-1) than in samples from the sub-epicardium (p=0.021) and the sub-endocardium (p=0.015). Non-failing mid-myocardial samples also produced more isometric force (14.3±1.33kNm-2) than samples from the sub-epicardium (p=0.008) and the sub-endocardium (p=0.026). Heart failure reduced power (p=0.009) and force (p=0.042) but affected the mid-myocardium more than the other transmural regions. Fibrosis increased with heart failure (p=0.021) and mid-myocardial tissue from failing hearts contained more collagen than matched sub-epicardial (p<0.001) and sub-endocardial (p=0.043) samples. Power output was correlated with the relative content of actin and troponin I, and was also statistically linked to the relative content and phosphorylation of desmin and myosin light chain-1.Non-failing human hearts exhibit transmural heterogeneity of contractile properties. In failing organs, region-specific fibrosis produces the greatest contractile deficits in the mid-myocardium. Targeting fibrosis and sarcomeric proteins in the mid-myocardium may be particularly effective therapies for heart failure. © 2014 Elsevier Ltd.