Developmental changes in contractile behavior are known to occur during fetal and postnatal heart development. In this study, we examined whether adaptations take place in titin. A range of species was used to evaluate titin isoform expression and altered function during cardiac muscle development. A novel titin exon microarray that allows all 363 titin exons to be monitored simultaneously was used for transcript studies. Results reveal expression of fetal titin isoforms, characterized by additional spring elements both in the tandem Ig and PEVK region of the molecule. At the protein level, the fetal cardiac isoform predominates in fetal and neonatal myocardium and gradually disappears during postnatal development with a time course that varies in different species. Passive myocardium, contrary to previous reports, was found to be less stiff in the neonate than in the adult. This can be explained by the unique spring composition of fetal cardiac titin expressed by the neonate. Changes in titin expression are likely to impact functional transitions and diastolic filling behavior during development of the heart.