The giant protein titin (3–3.7 MDa) is expressed in two main isoforms in the heart: small and stiff N2B and large and compliant N2BA isoforms. During development the hearts of rats express a fetal N2BA isoform (~3.7 MDa) but no N2B. After birth, the fetal N2BA is replaced by smaller-size N2BA-isoforms and N2B, which predominates in adult hearts. Here we show that this kind of perinatal titin-isoform switch does not occur in the hearts of guinea pig (GP) and sheep, in which N2B and N2BA are highly expressed in embryonic stages. By selective proteolysis of titin in skinned cardiac-fiber bundles we measured its contribution to passive tension (PT), compared to that of extracellular matrix (ECM) proteins. Titin-based PT contributed similarly to total PT, 45–58%, in adult sheep, GP and rat. In fetal GP and sheep, the contribution of titin to PT also reached 40–50%, whereas in fetal rat only 20% of the PT was titin-based. In rats, the switching of titin-isoforms is correlated to a switch in Troponin-I (TnI) isoform expression, where fetal ssTnI is replaced by cardiac cTnI shortly after birth, thereby reducing Ca²⁺-sensitivity. This shift in Ca²⁺-sensitivity is almost absent in GP where ssTnI and cTnI are co-expressed in fetal hearts. We conclude (i) that TnI and titin-isoform switching is a genetically programmed event and (ii) that titin- and ECM-based PT changes during development may counterbalance one another in a coordinated fashion.