It is generally accepted that abnormal intracellular Ca²⁺ handling accounts for the depressed left ventricular (LV) systolic and diastolic function in failing hearts. These findings have sparked a number of experimental and clinical studies aimed at restoring intracellular Ca²⁺ homoeostasis using viral gene transfer of Ca²⁺ transporter to the human myocardium; however, significant abnormalities in Ca²⁺ handling characterise late-stage heart failure and may not be the initiating factor. To test this hypothesis, we compared LV myocytes from C57BL6 mice 12 weeks after coronary ligation (causing an infarct >35% by 3D echocardiography) or sham surgery. LV ejection fraction was significantly reduced in infarcted mice versus shams (23% vs 55%, p<0.0001, n=9 in each group) and the heart weight or lung weight to body weight ratios were significantly increased. LV myocytes isolated from infarcted hearts were significantly larger than those from sham operated hearts (p=0.001). The distribution of cell shortening and cell size measured in ‘unselected’ LV myocytes (ie, from all the live cells in a given field) was much wider in infarcted mice than in sham-operated controls. However, basal cell shortening (3 Hz, field stimulation at 35°C) was enhanced in myocytes from infarcted hearts (p=0.001), as was the amplitude of the [Ca²⁺]_i transient (p<0.0001) and the SR Ca content (10 mM caffeine spritz, p=0.04). The rate of decay of [Ca²⁺]_i was faster both in field-stimulated (p<0.0001) and caffeine-induced [Ca²⁺]_i transients, suggesting upregulation of the Na⁺/Ca²⁺ exchanger activity. The myocyte inotropic response to increasing doses of isoproterenol or stimulation frequency (0.5–3 Hz) was similar, despite significant LV dysfunction and reduced β-adrenergic reserve in vivo. In summary, impaired LV performance post-myocardial infarction can occur in the absence of obvious Ca²⁺ and contraction abnormalities at the single myocyte level. These findings suggest that relatively minor changes in myocardial architecture, in terms of the distribution of cell properties, may lead to significant LV dysfunction and to the syndrome of heart failure well before individual myocytes show a significant deficit in contractility.