Passive myocardial stiffness is an important variable for the regulation of ventricular relaxation, and is defined by two main components: the rigidity of the collagenous connective tissue, and the stiffness of the myofilaments. The latter is determined by the giant protein titin, which spans a half-sarcomere from the Z-disk to M-line. In mammalian heart titin is expressed in two main isoform types: the shorter, stiff N2B isoform (3.0 MDa) and the longer, more compliant N2BA isoforms (> 3.2 MDa). During fetal heart development the expression ratio shifts in favor of the N2B isoform, which leads to increased titin-based myocardial stiffness. This process can be partly reversed in end-stage failing hearts, resulting in elevated levels of compliant N2BA-titin and reduced titin-stiffness. The composition of titin isoforms is hormonally regulated. In cell cultures of fetal rat cardiomyocytes the thyroid hormone triiodo-L-thyronine and insulin promote the expression of stiffer N2B-titin through activation of the phosphoinositide 3-kinase pathway. Through another, NO/cGMP-dependent pathway insulin is involved in enhanced phosphorylation of titin. Previous studies demonstrated that phosphorylation of the extensible titin N2-B unique sequence by cAMP- or cGMP-dependent protein kinase reduces myofilament stiffness, whereas phosphorylation of the spring-like PEVK domain by protein kinase C increases it. Our recent findings show that coordinated changes in the phosphorylation status of titin domains increases myofilament stiffness in failing hearts. We therefore conclude that titin-mediated myocardial stiffness is dynamically regulated by differential isoform expression and posttranslational modifications, and that malfunction of the hormonal system could play an important role in the development of diastolic dysfunction.