β-adrenergic stimulation in heart leads to increased contractility and lusitropy via activation of protein kinase A (PKA). In the cardiac sarcomere, both myosin binding protein C (cMyBP-C) and troponin-I (cTnI) are prominent myofilament targets of PKA. Treatment of permeabilized myocardium with PKA induces enhanced myofilament length dependent activation (LDA), the cellular basis of the Frank-Starling cardiac regulatory mechanism. It is not known, however, which of these targets mediates the altered LDA, and to what extent. Here, we employed two genetic mouse models in which the three PKA sites in cMyBP-C were replaced with either phospho-mimic (DDD) or phospho-null (AAA) residues. AAA or DDD permeabilized myocytes (n=12-17) were exchanged (~93%) for recombinant cTnI in which the two PKA sites were mutated to either phospho-mimic (DD) or phospho-null (AA) residues. Force-[Ca2+] relationships were determined at two sarcomere lengths (SL=1.9 um and SL=2.3 um). Data were fit to a modified Hill equation for each individual cell preparation at each SL. LDA was indexed as ΔEC50, the difference in [Ca2+] required to achieve 50% force activation at the two SL. We found that PKA mediated phosphorylation of cMyBP-C and cTnI each independently contribute to enhance myofilament length-dependent activation properties of the cardiac sarcomere, with relative contributions of ~67% and ~33% for cMyBP-C for cTnI, respectively. We conclude that β-adrenergic stimulation enhances the Frank-Starling regulatory mechanism predominantly via cMyBP-C PKA mediated phosphorylation. We speculate that this molecular mechanism enhances cross-bridge formation at long SL, while accelerating cross-bridge detachment and relaxation at short SL.