Phosphorylation of myosin regulatory light chain (RLC) by myosin light chain kinase (MLCK)and myosin binding protein-C (cMyBP-C) by protein kinase A (PKA) independently accelerate the kinetics of force development in ventricular myocardium. However, while MLCK treatment has been shown to increase the Ca 2+ sensitivity of force (pCa 50 ), PKA treatment has been shown to decrease pCa 50 , presumably due to cardiac troponin I phosphorylation. Further, MLCK treatment increases Ca 2+ -independent force and maximum Ca 2+ -activated force, whereas PKA treatment has no effect on either force. To investigate the structural basis underlying the kinase-specific differential effects on steady-state force, we used synchrotron low-angle X-ray diffraction to compare equatorial intensity ratios (I 1,1 /I 1,0 ) to assess the proximity of myosin cross-bridge mass relative to actin and to compare lattice spacings (d 1,0 ) to assess the inter-thick filament spacing in skinned myocardium following treatment with either MLCK or PKA. As we showed previously, PKA phosphorylation of cMyBP-C increases I 1,1 /I 1,0 and, as hypothesized, treatment with MLCK also increased I 1,1 /I 1,0 , which can explain the accelerated rates of force development during activation. Importantly, interfilament spacing was reduced by ∼2 nm ( 3.5%) with MLCK treatment, but did not change with PKA treatment. Thus, RLC or cMyBP-C phosphorylation increases the proximity of cross-bridges to actin, but only RLC phosphorylation affects lattice spacing, which suggests that RLC and cMyBP-C modulate the kinetics of force development by similar structural mechanisms; however, the effect of RLC phosphorylation to increase the Ca 2+ sensitivity of force is mediated by a distinct mechanism, most probably involving changes in interfilament spacing.