A long-term goal of work in our laboratories is to understand the structural basis of myofilament function, i.e. cross-bridge dynamics (CD) and myofilament length dependent activation (LDA) in striated muscle in heath and disease. Moreover, in cardiac muscle LDA underlies the Frank Starling Law of the Heart. Troponin phosphorylation, and in particular troponin-I (cTnI), has been suggested to be a pivotal modulator of myofilament function. Here we examined the impact of phosphorylation of distinct cTnI domains on CD and LDA in isolated human myocardium. Site specific phosphorylation was accomplished by charge mutation of hcTnI phosphomimics on the PKA (S23/24D), PKC (S42/44E; T143E), AMPK (S150D), and novel (S5/6D) sites, followed by recombinant protein exchange into skinned non-failing human LV cardiac muscle strips (∼2mm long, and ∼150um diameter. Force and ATPase activity was measured as function of [Ca2+] at short and long sarcomere length (SL=2.0&2.3um). We found, LDA: hcTnI-S150D attenuated, hcTnI-S42/43E increase, and no effect for the other sites. CD as indexed by tension cost was: decreased for cTnI-S42/44E and hcTnI-S150D, and no effect for the other sites. We conclude that cTnI phosphorylation at distinct sites differentially affect cross-bridge cycling and length dependent activation in human myocardium. Structural analysis employing x-ray diffraction is underway to determine the structural basis for these phenomena. Supported by NIH HL075494, HL62426, GM103622.