During cardiac filling, the relative contribution of distension of interstitial collagen, of distension of cytoskeletal proteins, and of low-grade diastolic cross-bridge cycling in the generation of diastolic left ventricular (LV) pressure remains unclear. In failing myocardium, interstitial collagen deposition and cross-linking are augmented. This increase in collagen deposition is accompanied by expression of a stiffer isoform of titin in the cardiomyocytes. Higher diastolic stiffness of failing myocardium is therefore not necessarily related to increased interstitial collagen content. Moreover, phosporylation of titin by protein kinase A and G, and inhibition of titin-actin interaction by cytosolic calcium allow for dynamic modulation of its diastolic tension generation and could account for acute shifts of myocardial distensibility. Acute shifts of myocardial distensibility, as occur in hypertrophy or in demand ischemia, have usually been attributed to a diastolic resurgence of cross-bridge interaction. In hypertrophied and failing myocardium, the recent demonstrations of diastolic calcium release from the ryanodine receptor, of deficient diastolic calcium removal from the cytosol, and of enhanced myofilamentary calcium sensitivity support residual diastolic cross-bridge interaction. In demand ischemia, the role of calcium overload in the reduction of diastolic LV distensibility is less clear because of correction of the reduced diastolic LV distensibility by quick stretches but not by a calcium desensitizer. Simultaneous imposition in animal models of multiple molecular changes involving interstitial, cytoskeletal, and myofilamentary proteins could elucidate their relative importance for myocardial stiffness and lead to selective correction of diastolic LV dysfunction as a novel mode of heart-failure therapy.