Left ventricular diastolic dysfunction in patients with congestive heart failure is associated with significant morbidity and mortality and is characterised by impaired relaxation, increased myocardial stiffness and ventricular dilatation. In cardiac muscle, the cytoskeletal protein titin is responsible for the development of passive tension in the myocyte and is present as two alternatively spliced isoforms. The isoform ratio is thought to determine the passive tension in the myocyte (1). We wanted to characterise the mechanical properties of titin isoform populations by applying a tensile force to isolated titin molecules. Titin was extracted and purified from ferret left ventricular tissue (2). Tensile force was applied to isolated titin molecules by employing a molecular combing technique (3) whereby titin molecules partially adsorbed to a poly-l-lysine coated mica surface are exposed to forces of ~800pN by a receding meniscus. Visualisation of uncombed titin molecules by atomic force microscopy revealed a highly coiled, spring-like conformation. In contrast, combed molecules were straightened and aligned. Uncombed titin monomers ranged in length from 0.6 to 1.4μm which we suggest is due to the presence of splice variants. The mean contour lengths were increased following molecular combing (2.24±0.16µm uncombed vs 2.60 ±0.21µm combed, p<0.05 n=42) and axial height profiles of entire titin molecules revealed localized areas of thinning. Mean axial heights were reduced following combing (0.62±0.21nm uncombed vs 0.58±0.22nm p<0.001 n=4000 ). The molecular combing technique is capable of aligning and elongating titin molecules and would be appropriate for the determination of mechanical properties of titin in disease.