Myofilament length–dependent activation is the main cellular mechanism responsible for the Frank–Starling law of the heart. All striated muscle display length-dependent activation properties, but it is most pronounced in cardiac muscle and least in slow skeletal muscle. Cardiac muscle expressing slow skeletal troponin (ssTn)I instead of cardiac troponin (cTn)I displays reduced myofilament length–dependent activation. The inhibitory region of troponin (Tn)I differs by a single residue, proline at position 112 in ssTnI versus threonine at position 144 in cTnI. Here we tested whether this substitution was important for myofilament length–dependent activation; using recombinant techniques, we prepared wild-type cTnI, ssTnI, and 2 mutants: cTnI_Thr>Pro and ssTnI_Pro>Thr. Purified proteins were complexed with recombinant cardiac TnT/TnC and exchanged into skinned rat cardiac trabeculae. Force–Ca²⁺ relationships were determined to derive myofilament Ca²⁺ sensitivity (EC₅₀) at 2 sarcomere lengths: 2.0 and 2.2 µm (n=7). Myofilament length-dependent activation was indexed as EC₅₀, the difference in EC₅₀ between sarcomere lengths of 2.0 and 2.2 µm. Incorporation of ssTnI compared with cTnI into the cardiac sarcomere reduced EC₅₀ from 1.26±0.30 to 0.19±0.04 µmol/L. A similar reduction also could be observed when Tn contained cTnI_Thr>Pro (EC₅₀=0.24±0.04 µmol/L), whereas the presence of ssTnI_Pro>Thr increased EC₅₀ to 0.94±0.12 µmol/L. These results suggest that Thr144 in cardiac TnI modulates cardiac myofilament length–dependent activation.